AU2003284578B2 - Novel 2' ,5' -oligoadenylic acid analogues - Google Patents

Novel 2' ,5' -oligoadenylic acid analogues Download PDF

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AU2003284578B2
AU2003284578B2 AU2003284578A AU2003284578A AU2003284578B2 AU 2003284578 B2 AU2003284578 B2 AU 2003284578B2 AU 2003284578 A AU2003284578 A AU 2003284578A AU 2003284578 A AU2003284578 A AU 2003284578A AU 2003284578 B2 AU2003284578 B2 AU 2003284578B2
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Makoto Koizumi
Koji Morita
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Daiichi Sankyo Co Ltd
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Description

S- 1
DESCRIPTION
NOVEL 2',5'-OLIGOADENYLATE ANALOGS TECHNICAL FIELD The present invention relates to analogs of oligoadenylate (2-5A) that are stable and have superior activity (particularly antitumor activity).
BACKGROUND ART which is known as a biological substance that has antiviral activity (Pharmacol. Ther. Vol. 78, No. 2, pp. 55-113, 1998), is a short-chain oligonucleotide composed of three or more adenosine units in which two adenosine 2' and 5' hydroxyl groups are linked with phosphate 2',5'-phosphodiester bonds, and in which a triphosphate group is bonded to the 5' end. When cells infected by a virus are subjected to extracellular interferon stimulation, 2-5A synthetase is induced in the presence of viral dsRNA, and 2-5A is produced from ATP. 2-5A is a substance that converts the inactive form of the RNA degrading enzyme, RNase L, into the active form within host cells. This activated RNase L inhibits viral growth in cells by degrading viral RNA. Moreover, when ovarian cancer cells HeylB are transfected with 2-5A, sequence-specific cleavage of 18S rRNA is known to occur, that results in demonstration of antitumor activity as a result of apoptosis through release of cytochrome c and activation of caspase Interferon Cytokine Res., 1091-1100 (2000)). Thus, 2-5A is expected to act as a virus growth inhibitor, and, more specifically, as an antivirus drug or antitumor drug.
In an in vitro experiment, an oligonucleotide composed of three or more adenosine units having a monophosphate group on the 5' end and linked with phosphodiester bonds is known to activate RNase L (Pharmacol. Ther. Vol. 78, No. 2, pp. 55-113, 1998; J. Biol. Chem. Vol. 270, No. 11, pp. 5963-5978). However, itself is easily degraded to AMP and ATP by 2'phosphodiesterase and nuclease. Moreover, the S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 S- 2 group or 5'-triphosphate group ends up being dephosphorylated by phosphatases in the living body and losing activity. Thus, in the case of using 2-5A as a virus growth inhibitor or antitumor drug, a 2-5A analog is desirable that has similar activity, but has high stability, making it more resistant to degradation and metabolism in the living body.
In order to overcome these shortcomings, various methods have been attempted as examples of modifying the phosphate groups. Examples of known methods include a method in which the non-bridging oxygen atom bonded to the phosphorus atom of the phosphodiester bond of the oligonucleotide is substituted with a sulfur atom (phosphorothioate modification), a method in which said oxygen atom is substituted with a methyl group, a method in which said oxygen atom is substituted with a boron atom, and a method in which the sugar portion or nucleobase portion of the oligonucleotide is chemically modified (Freier, Altmann, Nucleic Acids Res., 25, 4429 (1997)). A known example of such a 2-5A analog is the adenosine tetramer which has undergone the phosphorothioate modification shown below (Carpten, J. et al.
Nature Genetics, 30, 181 (2002)).
NH
2 Ns S N N OH O NH 2 OH 0 N' 'NJ S=P-OH N N
NH
2 OH 0 S=P-OH N N
NH
2 OH O N 'N S=P-OH N N OH OH Moreover, analogs having a chemical structure like that shown below, in which the sugar portion of adenosine has been S:/ChemicalSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 S-3 modified, are described in Japanese Patent Application (Kokai) No. Hei 10-195098 and Japanese Patent No. 3420984 as adenosine units of 2-5A analogs.
NH
2 Y'ON N
A,
0 OY 2 (In the above formula, Y' and Y 2 represent a hydrogen atom or a protecting group for a hydroxy group, and A represents an alkylene group having from 1 to 3 carbon atoms.) In addition, a 2-5A molecule bonded by means of a linker with an antisense molecule in the form of an oligonucleotide having a sequence complementary to mRNA involved in diseases has been used as a 2-5A antisense oligonucleotide that inhibits the function of mRNA Adahet, et al., Current Medicinal Chemistry (2001), 8, 1189-1212). A highly stable 2-5A analog that is resistant to degradation and metabolism in the living body serves as a portion of a superior 2-5A antisense oligonucleotide, and is expected to be a useful drug. In particular, oligonucleotides containing a bridged nucleoside in which an oxygen atom at the 2' position and a carbon atom at the 4' position of the sugar portion are bonded with an alkylene group are known to be useful as antisense molecules (Japanese Patent Application (Kokai) No. Hei 10-304889, Japanese Patent Application (Kokai) No. 2000-297097).
DISCLOSURE OF THE INVENTION The inventors of the present invention conducted extensive research over the course of many years on non-natural type 2-5A analogs that have antivirus activity, antitumor activity or superior antisense activity, are stable in the living body, and are associated with the occurrence of few adverse side effects. As a result, they were found to be useful as stable and superior antivirus drugs, antitumor drugs and S :/hemicaUSankyo/FP00343/FP200343s .doc P89346/FP-200343/gds-mg/specification//O3.05,05 O antisense drugs, thereby leading to completion of the present invention.
The 2-5A analog of the present invention relates to a 00 2 ',5'-oligoadenylate analog represented by the general formula 0 0 0 0 0 0 O ii 1 11 II231 i RI-p-E -p-E-P-E3 P-E -R R
SR
2
R
3
R
4 R R tn m n C [wherein m represents 0; n represents 0 or 1; R 1 represents an alkoxy group having from 1 to 6 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, an alkylthio group having from 1 to 4 carbon atoms which may be substituted, an amino group, an amino group protected by a nucleic acid synthesis protecting group, an amino group substituted by alkyl group(s) having from 1 to 6 carbon atoms which may be substituted, an alkyl group having from 1 to 6 carbon atoms which may be substituted, an aryloxy group which may be substituted, or an arylthio group which may be substituted, or a group of formula: XI-X 2
-X
3
R
2
R
3
R
4
R
5 and R 6 represent a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, an alkoxy group having from 1 to 4 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, an alkylthio group having from 1 to 4 carbon atoms which may be substituted, or a group of formula: Xi-Xz-X 3
R
7 represents an oxygen atom, a sulfur atom, a -O(CH 2
CH
2 0)qgroup (q represents an integer of 2 to or an oxyalkyleneoxy group having from 1 to 6 carbon atoms; R 8 represents a hydrogen atom, an alkyl group having from 1 to 6 carbon atoms which may be substituted, an aralkyl group which may be substituted, an aryl group which may be substituted, or a oligonucleotide analog which has one hydroxyl group removed from the 5'-phosphoric acid group; El represents K 2
E
2 represents K 1
E
3 represents K 2 or K 3 and E' represents K 1
K
2
K
3 or K 4
K
2 0 K 3 and K 4 represent OH 0 OD 0 0 0 0 0 0 K
K
2
K
3
K
4 k respectively, wherein, B represents a purin-9-yl group or a 0 substituted purin-9-yl group having substituent(s) selected from Sthe following Group a, A represents an alkylene group having Sfrom 1 to 4 carbon atoms, D represents an alkyl group having from 1 to 6 carbon atoms which may be substituted, or an alkenyl group having from 2 to 6 carbon atoms which may be substituted); XI represents an alkyl group having from 1 to 24 carbon atoms which may be substituted, or an aryl group which may be substituted, or an aralkyl group which may be substituted;
X
2 represents a -NHC(=O)NH-, or a group; and X 3 represents an alkylene group having from 1 to 6 carbon atoms which may be substituted] (provided that compounds in which n is 1, R 2
R
3
R
4 and R 6 are a hydroxyl group, R 7 is an oxygen atom, and R e is a 2hydroxyethyl group are excluded), or a pharmacologically acceptable salt thereof.
(Group a) a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, an alkoxy group having from 1 to 6 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, an alkylthio group having from 1 to 4 carbon atoms which may be substituted, an amino group, 0 an amino group protected by a nucleic acid synthesis protecting group, an amino group substituted by alkyl group(s) having from 00 1 to 4 carbon atoms which may be substituted, an alkyl group having from 1 to 6 carbon atoms which may be substituted, and 00 r- a halogen atom.
kn 'I The above 2',5'-oligoadenylate analog or 00 (C pharmacologically acceptable salt thereof is preferably S(1) a 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof, in which R 1 is an alkoxy group having from 1 to 4 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, or an alkylthio group having from 1 to 4 carbon atoms which may be substituted, or a group of formula: Xi-X 2
-X
3 Xi is an alkyl group having from 10 to 24 carbon atoms which may be substituted; X 2 is a or group; and X 3 is an alkylene group having from 1 to 4 carbon atoms which may be substituted; a 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof, in which R 7 represents an oxygen atom, a -O(CH 2
CH
2 0)q- group (q represents an integer of 2 to or an oxyalkyleneoxy group having from 1 to 6 carbon atoms; and R 8 is a hydrogen atom;, an alkyl group having from 1 to 6 carbon atoms which may be substituted, or a 5'-phosphorylated oligonucleotide analog which has one hydroxyl group removed from the phosphoric acid group; a 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof, wherein D is a methyl group or a 2propenyl group; P:\OPER\PDB\Spec\2C0032B4578 2spa.doc 7/6/07 a 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof, wherein E 3 is K 3 and A is a methylene, ethylene, or propylene group; 00 a 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof, wherein B is a 6-aminopurin-9-yl (that 00 is, adeninyl), 6-amino-8-bromopurin-9-yl, 6-amino-8-chloropurin- 9-yl, 6-amino-8-fluoropurin-9-yl, 6-amino-8-methoxypurin-9-yl, 6-amino-8-ethoxypurin-9-yl, 6-amino-8-t-butoxypurin-9-yl, 6- 0C amino-2-bromopurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2- Sfluoropurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2- C( ethoxypurin-9-yl, 6-amino-2-t-butoxypurin-9-yl, or 2,6diaminopurin-9-yl group; or a 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof, wherein B is 6-aminopurin-9-yl (that is, adeninyl) or 6-amino-8-bromopurin-9-yl.
The invention also provides the use of a oligoadenylate analog or pharmacologically acceptable salt thereof, as claimed herein, in the preparation of an antitumour, antisense or antiviral medicament, as well as a method of treating a viral disease, a tumour or a disease that can be treated by antisense therapy comprising the administration of a analog or pharmalogically acceptable salt thereof, as claimed herein, to a subject in need thereof.
In the above general formula, the "alkylene group having from 1 to 4 carbon atoms" of A can be, for example, a methylene, ethylene, trimethylene or tetramethylene group, and is preferably an ethylene or trimethylene group.
In the above general formula the protecting group of the "hydroxyl group protected by a nucleic acid synthesis protecting group" of R 2
R
3 R and R 6 or the Group o is not particularly limited so long as it can stably protect a hydroxyl group during nucleic acid synthesis, and specifically means a protecting group stable under acidic or neutral conditions, and P:\OPER\PDB\Spec!\203328457S Zspa.doc 71/6/07 -8cleavable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis or photolysis. Such a protecting group can be, for example, an "aliphatic acyl group" such as an alkylcarbonyl group, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3ethyloctanoyl, 3, 7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1methylpentadecanoyl, 14-methylpentadecan oyl, 13, 13dimethyltetradecanoyl, heptadecanoyl, octadecanoyl, l-methylheptadecanoyl, nonadecanoyl, eicosanoyl and heneicosanoyl; a carboxylated alkylcarbonyl group, e.g., succinoyl, glutaroyl and adipoyl; a halogeno lower alkylcarbonyl group, chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl; a lower alkoxy lower alkylcarbonyl group, e.g., methoxyacetyl; or an unsaturated alkylcarbonyl group, 2-methyl-2-butenoyl; a "lower alkyl group" such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2methylbutyl, neopentyl, 1-ethyipropyl, n-hexyl, isohexyl, 4-.
methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3, 3-dimethylbutyl, 2, 2-dimethylbutyl, 1, 1-dimethylbutyl, 1,2dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl and 2ethylbutyl; a "lower alkenyl group" such as ethenyl, 1-propenyl, 2-propenyl, l-methyl-2-propenyl, l-methyl-l-propenyl, 2-methyl-l-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, l-methyl-2-butenyl, l-methyl-l-butenyl, 3-methyl-2-butenyl, 1ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3butenyl, l-ethyl-3-butenyl, 1-pentenyl, 2-pent enyl, l-methyl-2pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, l-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-pentenyl, 2-methyl- 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and hexenyl; an "aromatic acyl group" such as an arylcarbonyl group, e.g., benzoyl, at-naphthoyl and j-naphthoyl; a halogeno arylcarbonyl S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 9group, 2-bromobenzoyl and 4-chlorobenzoyl; a lower alkylated arylcarbonyl group, 2,4,6-trimethylbenzoyl and 4-toluoyl; a lower alkoxylated arylcarbonyl group, 4anisoyl; a carboxylated arylcarbonyl group, 2carboxybenzoyl, 3-carboxybenzoyl and 4-carboxy benzoyl; a nitrated arylcarbonyl group, 4-nitrobenzoyl and 2nitrobenzoyl; a lower alkoxycarbonylated arylcarbonyl group, 2-(methoxycarbonyl)benzoyl; or an arylated arylcarbonyl group, 4-phenylbenzoyl; a "tetrahydropyranyl or tetrahydrothiopyranyl group" such as tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4methoxytetrahydropyran-4-yl, tetrahydrothiopyran-2-yl and 4methoxytetrahydrothiopyran-4 -yl; a "tetrahydrofuranyl or tetrahydrothiofuranyl group" such as tetrahydrofuran-2-yl and tetrahydrothiofuran-2-yl; a "silyl group" such as a tni-lower alkylsilyl group, e.g., trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, tbutyldimethylsilyl, methyliisopropylsilyl, methyldi-tbutylsilyl and triisopropylsilyl; or a tni-lower alkylsilyl group substituted by 1 or 2 aryl groups, e.g., diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl and phenyldiisopropylsilyl; a "lower alkoxymethyl group" such as methoxymethyl, 1,1dimethyl -1-methoxymethyl, ethoxymethyl, propoxymethyl, i sopropoxymethyl, butoxymethyl and t-butoxymethyl; a "lower alkoxylated lower alkoxymethyl group" such as 2methoxyethoxymethyl; a "halogeno lower alkoxymethyl" such as 2,2,2trichioroethoxymethyl and bis (2-chloroethoxy)methyl; a "lower alkoxylated ethyl group" such as 1-ethoxyethyl and 1- (isopropoxy) ethyl; a "halogenated ethyl group" such as 2,2,2-trichloroethyl; a "methyl group substituted by from 1 to 3 aryl groups" such as benzyl, c-naphthylmethyl, P-naphthylmethyl, diphenylmethyl, triphenylmethyl, a-naphthyldiphenylmethyl and 9-anthrylmethyl; a "methyl group substituted by from 1 to 3 aryl groups whose SJ/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.O5 aryl ring is substituted by lower alkyl, lower alkoxy, halogen or cyano group(s)" such as 4-methylbenzyl, 2,4,6trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4methoxyphenyldiphenylmethyl, 4,4' -dimethoxytriphenylmethyl, 2nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl and 4- 00 cyanobenzyl; a "lower alkoxycarbonyl group" such as methoxycarbonyl, 00 ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl; an "aryl group substituted by halogen atom(s), lower alkoxy group(s) or nitro group(s)" such as 4-chlorophenyl, 2chlorophenyl, 4-methoxyphenyl, 4-nitrophenyl and 2,4cinitrophenyl; a "lower alkoxycarbonyl group substituted by halogen or trilower alkylsilyl group(s)" such as 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl; an "alkenyloxycarbonyl group" such as vinyloxycarbonyl and allyloxycarbonyl; an "aralkyloxycarbonyl group whose aryl ring may be substituted by 1 or 2 lower alkoxy or nitro groups" such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl and 4nitrobenzyloxycarbonyl; an "aliphatic acyloxymethyl group" such as an alkylcarbonyloxymethyl group, acetyloxymethyl, propionyloxymethyl, butyryloxymethyl, isobutyryloxymethyl, pentanoyloxymethyl, pivaloyloxymethyl, valeryloxymethyl, isovaleryloxymethyl, octanoyloxymethyl, nonanoyloxymethyl, decanoyloxymethyl, 3-methylnonanoyloxymethyl, 8methylnonanoyloxymethyl, 3-ethyloctanoyloxymethyl, 3,7dimethyloctanoyloxymethyl, undecanoyloxymethyl, dodecanoyloxymethyl, tridecanoyloxymethyl, tetradecanoyloxymethyl, pentadecanoyloxymethyl, hexadecanoyloxymethyl, 1-methylpentadecanoyloxymethyl, 14methylpentadecanoyloxymethyl, 13, 13dimethyltetradecanoyloxymethyl, heptadecanoyloxymethyl, methylhexadecanoyloxymethyl, octadecanoyloxymethyl, 1- P:\OPER\PDB\Speci\2003284578 1spa.doc 7/7/05 11 .methylheptadecanoyloxymethyl, nonadecanoyloxymethyl, eicosanoyloxymethyl and heneicosanoyloxymethyl; a carboxylated alkylcarbonyloxymethyl group, succinoyloxymethyl, glutaroyloxyrnethyl and adipoyloxymethyl; a halogeno lower alkylcarbonyloxymethyl group, chloroacetyloxymethyl, dichioroacetyloxynethyl, trichioroacetyloxymethyl and trifluoroacetyloxymethyl; a lower alkoxy lower alkylcarbonyloxymethyl group, methoxyacetyloxymethyl; or an unsaturated alkylca. bonyloxynethyl group, (E)-2-methyl- 2-butenoyl; an "aliphatic acylthioethyl group" such as an alkylcarbonylthioethyl group, acetylthioethyl, propionylthioethyl, butyrylthioethyl, isobutyrylthioethyl, pentanoylthioethyl, pivaloylthioethyl, valerylthioethyl, isovalerylthioethyl, octanoylthioethyl, nonanoylthioethyl, decanoylthioethyl, 3-iethylnonanoylthioethyl, 8methylnonanoylthioethyl, 3-ethyloctanoylthioethyl, 3,7dimethyloctanoylthioethyl, undecanoylthioethyl, dodecanoylthioethyl, tridecanoylthioethyl, tetradecanoylthioethyl, pentadecanoylthioethyl, hexadecanoylthioethyl, l-methylpentadecanoylthioethyl, 14methylpentadecanoylthioethyl, 13, 13direthyltetradecanoylthioethyl, heptadecanoylthioethyl, methylhexadecanoylthioethyl, octadecanoylthioethyl, 1iethylheptadecanoylthioethyl, nonadecanoylthioethyl, eicosanoylthioethyl and heneicosanoylthioethyl; a carboxylated alkylcarbonylthioethyl group, succinoylthioethyl, glutaroylthioethyl and adipoylthioethyl; a halogeno lower alkylcarbonylthioethyl group, chloroacetylthioethyl, dichloroacetylthioethyl, trichloroacetylthioethyl and trifluoroacetylthioethyl;, a lower alkoxy lower alkylcarbonylthioethyl group, methoxyacetylthioethyl; or an unsaturated alkylcarbonylthioethyl group, (E)-2-methyl- 2-butenoyl.
The protecting group of the "hydroxyl group protected by a nucleic acid synthesis protecting group" of R 2 R 3 R 4 R 5 and S:/ChemicallSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specificalon//03.05.05 -12- R 6 or the Group cc is preferably a "methyl group substituted by from 1 to 3 aryl groups", an "aryl group substituted by halogen atom(s), lower alkoxy group(s) or nitro group(s)", a "lower alkyl group", a "lower alkenyl group", an "aliphatic acyloxymethyl group", or an "aliphatic acylthioethyl group", more preferably a benzyl group, a 2-chlorophenyl group, a 4chlorophenyl group, a 2-propenyl group, a pivaloyloxymethyl group, an acetylthioethyl group, or a pival'oylthioethyl.group.
In the above general form ula the "alkoxy group having from 1 to 6 carbon atoms which may be substituted" of R 1 R 3, R 4, R' or the Group a can be, for example, a "lower a lkyloxy group" such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, n-pentyloxy, isopentyloxy, 2methylbutoxy, neopentyloxy, l-ethylpropoxy, n-hexyloxy, isohexyloxy, 4-methylpentyloxy, 3-methylpentyloxy, 2methylpentyloxy, 1-methylpentyloxy, 3, 3-dimethylbutoxy, 2,2dimethylbutoxy, 1, 1-dimethylbutoxy, 1, 2-dimethylbutoxy, 1,3dimethylbutoxy, 2,3-dimethylbutoxy and 2-ethylbutoxy; a "lower alkyloxy group substituted by hydroxyl group(s)" such as 1-hydroxymethyloxy, 2-hydroxyethyloxy, 3-hydroxypropyloxy, 4hydroxybutyloxy, 2-hydroxypropyloxy, l-methyl-2--hydroxyethyloxy, 1-methyl-l-hydroxyethyloxy, 1, l-dimethyl-2-hydroxyethyloxy, 2hydroxybutyloxy, 3-hydroxybutyloxy, 1-methyl-3-hydroxypropyloxy and 2-methyl-3-hydroxypropyloxy; a "lower-alkyloxy group substituted by amino group(s)" such as 1-aminomethyloxy, 2-aminoethyloxy, 3-aminop~opyloxy, 4aminobutyloxy, 2-aminopropyloxy, 1-methyl-2-aminoethyloxy, 1methyl-l-aminoethyloxy, 1, l-dimethyl-1-aminoethyloxy, 2aminobutyloxy, 3-aminobutyloxy, l-methyl-3-aminopropyloxy and 2methyl-3 -aminopropyloxy; a "lower alkyloxy group substituted by alkoxy group(s)" such as 1-methoxymethyloxy, 2-methoxyethyloxy, 3-methoxypropyloxy, 4methoxybutyloxy, 2-methoxypropyloxy, l-methyl-2-methoxyethyloxy, 1-methyl-l-methoxyethyloxy, 1, 1-dimethyl-2-methoxyethyloxy, 2methoxybutyloxy, 3-methoxybutyloxy, l-methyl-3-methoxypropyloxy, 2-methyl-3-methoxypropyloxy, 1-ethoxymethyloxy, 2-ethoxyethyloxy, S:/ChemicaIUSankyoIFP200343fFP00343s.doc P89346/FP-200343/gds-mgspecificaioni/03.OS.05 13 3-ethoxypropyloxy, 4-ethoxybutyloxy, 2-ethoxypropyloxy, 1methyl-2-ethoxyethyloxy, 1-methyl-l-ethoxyethyloxy, 1,1dimethyl-2-ethoxyethyloxy, 2-ethoxybutyloxy, 3-ethoxybutyloxy, l-methyl-3-ethoxypropyloxy and 2-methyl-3-ethoxypropyloxy; or a "cycloalkyloxy group" such as cyclopropoxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, norbornyloxy and adamantyloxy; and is preferably a 2-hydroxyethoxy group.
In the above general formula the "oxyalkyleneoxy group having from 1 to 6 carbon atoms" of R 7 can be, for example, an oxymethyleneoxy, oxyethyleneoxy, oxytrimethyleneoxy, oxytetramethyleneoxy, oxypentamethyleneoxy, or oxyhexamethyleneoxy group, and is preferably an oxytetramethyleneoxy or oxypentamethyleneoxy group.
In the above general formula the protecting group of the "mercapto group protected by a nucleic acid synthesis protecting group" of R R 2
R
3
R
4
R
5 and R 6 or the Group a is' not particularly limited so long as it can stably protect a mercapto group during nucleic acid synthesis, and specifically means a protecting group stable under acidic or neutral conditions, and cleavable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis or photolysis. Such a protecting group can be, for example, a "group which can form a disulfide" such as an alkylthio group, methylthio, ethylthio and tert-butylthio, or an arylthio group, e.g.
benzylthio, in addition to the groups listed as a protecting group of a hydroxyl group, and is preferably an "aliphatic acyl group", an "aromatic acyl group", an "aliphatic acyloxymethyl group", or an "aliphatic acylthioethyl group", more preferably a pivaloyloxymethyl group, an acetylthioethyl group, or a pivaloylthioethyl group.
In the above general formula the "alkylthio group having from 1 to 4 carbon atoms which may be substituted" of R 1
R
2
R
3 R R 5 and R 6 or the Group a can be, for example, methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, or tert-butylthio, and is preferably a methylthio or ethylthio group.
S:/ChemicaUSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 14 In the above general formula the protecting group of the "amino group protected by a nucleic acid synthesis protecting group" of R R R R and R 6 or the Group a is not particularly limited so long as it can stably protect an amino group during nucleic acid synthesis, and specifically means a protecting group stable under acidic or neutral conditions and cleavable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis or photolysis. Such a protecting group can be, for example, an "aliphatic acyl group" such as an alkylcarbonyl group, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1methylpentadecanoyl, 14-methylpentadecanoyl, 13,13dimethyltetradecanoyl, heptadecanoyl, octadecanoyl, l-methylheptadecanoyl, nonadecanoyl, eicosanoyl and heneicosanoyl; a carboxylated alkylcarbonyl group, e.g., succinoyl, glutaroyl and adipoyl; a halogeno lower alkylcarbonyl group, e.g.,.chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl; a lower alkoxy lower alkylcarbonyl group, e.g., methoxyacetyl; or an unsaturated alkylcarbonyl group, 2-methyl-2-butenoyl; an "aromatic acyl group" such as an arylcarbonyl group, benzoyl, a-naphthoyl and P-naphthoyl; a halogeno arylcarbonyl group, 2-bromobenzoyl and 4-chlorobenzoyl; a lower alkylated arylcarbonyl group, 2,4,6-trimethylbenzoyl and 4-toluoyl; a lower alkoxylated arylcarbonyl group, 4anisoyl; a carboxylated arylcarbonyl group, 2carboxybenzoyl, 3-carboxybenzoyl and 4-carboxybenzoyl; a nitrated arylcarbonyl group, 4-nitrobenzoyl and 2nitrobenzoyl; .a lower alkoxycarbonylated arylcarbonyl group, 2-(methoxycarbonyl)benzoyl; or an arylated arylcarbonyl group, 4-phenylbenzoyl; a "lower alkoxycarbonyl group" such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl; S:/ChemicalSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification/03.05.05 c a "lower alkoxycarbonyl group substituted by halogen or trilower alkylsilyl group(s)" such as 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl; C an "alkenyloxycarbonyl group" such as vinyloxycarbonyl and allyloxycarbonyl; or 00 an "aralkyloxycarbonyl group whose aryl ring may be substituted V by 1 or 2 lower alkoxy or nitro groups" such as 00 benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl and 4nitrobenzyloxycarbonyl; and is preferably an "aliphatic acyl CA group" or an "aromatic acyl group", more preferably a benzoyl group.
In the above general formula the "amino group substituted by alkyl group(s) having from 1 to 4 carbon atoms which may be substituted" of R1, R2 R R, R 5 and R 6 or the Group a can be, for example, a "lower alkylamino group" such as methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, s-butylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di(s-butyl)amino and di(tertbutyl)amino; a "lower alkylamino group substituted by hydroxyl group(s), lower alkoxy group(s) or halogen atom(s)" such as 1hydroxyethylamino, 2-hydroxyethylamino, 1-methoxyethylamino, 2methoxyethylamino, 1-bromoethylamino, 2-methoxyethylamino, 1chloroethylamino and 2-chloroethylamino; or a "lower alkoxycarbonylamino group" such as 1methoxycarbonylethylamino, 2-methoxycarbonylethylamino, 1ethoxycarbonylethylamino, 2-ethoxycarbonylethylamino, 1propoxycarbonylethylamino and 1-propoxycarbonylethylamino; and is preferably a 1-hydroxyethylamino, 2-hydroxyethylamino, methylamino, ethylamino, dimethylamino, diethylamino, diisopropylamino, 1-methoxycarbonylethylamino or 1ethoxycarbonylethylamino group.
In the above general formula the "alkyl group having from 1 to 6 carbon atoms which may be substituted" of D, R R2, R R R R8 or the Group a can be, for example, a P:\OPER\PD\Speci\2003284578 1spa.doc 7/7/05 0 -16- "lower alkyl group" such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, .2methylbutyl, neopentyl, 1-ethyipropyl, n-hexyl, isohexyl, 4methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3dimethylbutyl, dimethylbuy 2,3-dimethylbut yl and 2ethylbutyl; a "lower alkyl group substituted by hydroxyl group(s)" such as 1-hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 2-hydroxypropyl, l-methyl-2-hydroxyethyl, l-methyl-lhydroxyethyl, 1, 1-dimethyl-2-hydroxyethyl, 2-hydroxybutyl, 3hydroxybutyl, l-methyl-3-hydroxypropyl and 2-methyl-3-.
hydroxypropyl; a "lower alkyl group substituted by amino group(s)" such as 1aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 2aminopropyl, l-methyl-2--aminoethyl, l-methyl-l-aminoethyl, 1,1dimethyl-2-aminoethyl, 2-aminobutyl, 3-aminobutyl, 1-methyl-3aminopropyl and 2-methyl-3-aminopropyl; a "lower alkyl group substituted by alkoxy group(s)" such as 1met hoxymethyl, 2-methoxyethyl, 3-methoxypropyl, 4-methoxybutyl, 2-methoxypropyl, l-methyl-2-methoxyethyl, 1-methyl-lmethoxyethyl, 1, 1-dimethyl-2-methoxyethyl, 2-methoxybutyl, 3methoxybutyl, 1-methyl-3-methoxypropyl, 2-methyl-3-methoxypropyl, 1-ethoxymethyl, 2-ethoxyethyl, 3-ethoxypropyl, 4-ethoxybutyl, 2ethoxypropyl, 1-methyl-2-ethoxyethyl, l-methyl-l-ethoxyethyl, 1, 1-dimethyl-2-ethoxyethyl, 2-ethoxybutyl, 3-ethoxybutyl, 1methyl-3-ethoxypropyl and 2-methyl-3-ethoxypropyl; or.
a "cycloalkyl group" such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl and adamantyl; and is preferably a 2-methoxyethyl group or a 2-hydroxyethyl group.
In the above general formula the "alkyl group having from 1 to 24 carbon atoms which may be substituted" of X, can be, for example, stearyl, 2,2-dimethylstearyl, heptadecyl, 2, 2-dimethylheptadecyl, hexadecyl, 2, 2-dimethylhexadecyl, pentadecyl, 2, 2-dimethylpentadecyl, tetradecyl, 2,2- S:/CheinicaIISankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 17 dimethyltetradecyl, tridecyl, 2,2-dimethyltridecyl, dodecyl, 2,2-dimethyldodecyl, undecyl, 2,2-dimethylundecyl, decyl, 2,2dimethyldecyl, nonyl, 2,2-dimethylnonyl, octyl, 2,2dimethyloctyl, heptyl, 2,2-dimethyiheptyl, hexyl, 2,2dimethylhexyl, pentyl, 2,2-dimethylpentyl, butyl, 2,2dimethylbutyl, propyl, 2,2-tert-butyl, ethyl, or methyl, and is preferably stearyl or 2,2-dimethylstearyl.
In the above general formula the "alkylene group having from 1 to 6 carbon atoms which may be substituted" of X 3 can be, for example, methylene, ethylene, propylene, butylene, 2,2-dimethylethylene, 2,2-dimethylpropylene, or 2,2dimethylbutylene, and is preferably methylene or ethylene.
In the above general formula the "aryloxy group which may be substituted" of R 1 can be, for example, an "aryloxy group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)" such as 2-methylphenoxy, 3-methylphenoxy, 4methylphenoxy, 2,6-dimethylphenoxy, 2-chlorophenoxy, 4chlorophenoxy, 2,4-dichlorophenoxy, 2,5-dichlorophenoxy, 2bromophenoxy, 4-nitrophenoxy and 4-chloro-2-nitrophenoxy.
In the above general formula the "aryl group which may be substituted" of R 8 or X 1 can be, for example, an "aryl group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)" such as 2-methylphenyl, 3-methylphenyl, 4methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2-bromophenyl, 4nitrophenyl and 4-chloro-2-nitrophenyl.
In the above general formula the "arylthio group which may be substituted" of R' can be, for example, an "arylthio group substituted by lower alkyl group(s), halogen atom(s) or nitro group(s)" such as 2-methylphenylthio, 3methylphenylthio, 4-methylphenylthio, 2,6-dimethylphenylthio, 2chlorophenylthio, 4-chlorophenylthio, 2,4-dichlorophenylthio, 2-bromophenylthio, 4-nitrophenylthio and 4-chloro-2-nitrophenylthio.
In the above general formula the "alkenyl group having from 2 to 6 carbon atoms which may be substituted" of D S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 18 can be, for example, ethenyl, 1-propenyl, 2-propenyl, 1-methyl- 2-propenyl, 1-methyl-l-propenyl, 2-methyl-l-propenyl, 2-methyl- 2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl- 2-butenyl, l-methyl-l-butenyl, 3-methyl-2-butenyl, 1-ethyl-2butenyl, 3-butenyl, l-methyl-3-butenyl, 2-methyl-3-butenyl, 1ethyl-3-butenyl, 1-pentenyl, 2-pentenyl, l-methyl-2-pentenyl, 2methyl-2-pentenyl, 3-pentenyl, l-methyl-3-pentenyl, 2-methyl-3pentenyl, 4-pentenyl, l-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or In the above general formula the "aralkyl group which may be substituted" of R 8 or X, can be, for example, an "aralkyl group" such as benzyl, a-naphthylmethyl, 3 naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl, triphenylmethyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, l-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, l-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, naphthylpentyl, 1-phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4phenylhexyl, 5-phenyihexyl, 6-phenylhexyl, 1-naphthylhexyl, 2naphthylhexyl, 3 -naphthylhexyl, 4 -naphthylhexyl, 5 -naphthylhexyl and 6-naphthylhexyl; or an "aralkyl group whose aryl ring is substituted by nitro group(s) or halogen atom(s)" such as 4chlorobenzyl, 2- (4-nitrophenyl) ethyl, o-nitrobenzyl, 4nitrobenzyl, 2, 4-dinitrobenzyl and 4-chloro-2-nitrobenzyl.
In the-above general formula of all of the "purin- 9-yl group" and the "substituted purin-9-yl group" of B, the preferred groups are 6-amino-purin-9-yl (that is, adeninyl), 6amino-purin-9-yl in which-the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-bromopurin-9yl, 6-amino-8-bromopurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino- B-chloropurin-9-yl, 6-amino-8-chloropurin-9-yl in which the S:/ChemicallSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specificatioi//03.S.0S 19 amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-fluoropurin-9-yl, 6-amino-8-fluoropurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-methoxypurin-9-yl, 6-amino-8methoxypurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-8-ethoxypurin- 9-yl, 6-amino-8-ethoxypurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino- 8-t-butoxypurin-9-yl, 6-amino-8-t-butoxypurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2amino-6-chloropurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 2-amino-6fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 2-amino-6-hydroxypurin-9-yl (that is, guaninyl), 2-amino-6-hydroxypurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6amino-2-methoxypurin-9-yl, 6-amino-2-methoxypurin-9-y in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-2-chloropurin-9-yl, 6-amino-2chloropurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 6-amino-2-fluoropurin- 9-yl, 6-amino-2-fluoropurin-9-yl in which the amino group is protected by a nucleic acid synthesis protecting group, 2,6dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, and 6mercaptopurin-9-yl group, and the more preferred groups are 6benzoylaminopurin-9-yl or adeninyl.
There is no particular limitation on the functional group represented by "X-X 2
-X
3 provided that it is a combination comprising X 1
X
2
X
3 and S mentioned above, and it can be, for example, an acyloxyalkylthio group such as 2- (stearoyloxy)ethylthio, 2-(myristoyloxy)ethylthio, 2- (decanoyloxy)ethylthio, 2-(benzoyloxy)ethylthio, 2- S:/ChemicalSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 20 (p ival oyloxy) ethyl thi o, 2 2 -dime thyloctade canoyl oxy) ethylithiao, 3- (stearoyloxy)propylthio, 3- (myristoyloxy)propylthio, 3- (decanoyloxy) propylthio, 3- (benzoyloxy) propylthio, 3- (pivaloyloxy)propylthio, 3- (2,2dimethyloctadecanoyloxy)propylthio, 4- (stearoyloxy)butylthio, 4- (myristoyloxy)butylthio, 4- (decanoyloxy)butylthio, 4- (benzoyloxy)butylthio, 4- (pivaloyloxy)butylthio and 4- (2,2dimethyloctadecanoyloxy) butyithia, or an alkylcarbamoyloxyalkylthio group such as 2- (stearylcarbamoyloxy)ethylthio, or the following compounds: 0 0
HIC
0
H
S
M
3 C 0II S
S
H3C O S 0 HC 0 0
H~H
0 FhC H O S
H~C
0 H~C H H
S
HC NI and is preferably a 2-stearoyloxyethylthio or 2-(2,2dimethyloctadecanoyloxy) ethylthio group.
In the above general formula the "halogen atom" of the Group a can be, for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and is preferably a bromine atom or a chlorine atom.
The '2',5'-oligoadenylate analog (2-5A analog)" means a non-natural type derivative of "2',5'-oligoadenylate", in which the 2' position and 5' position of the 3 or 4 "nucleosides", being the same or different, are bonded by a phosphodiester bond S:/ChemicallSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 S- 21linkage or a modified phosphodiester linkage, and a phosphoryl derivative is bonded to the 5'-terminal, or a phosphoryl derivative is optionally bonded to the 2'-terminal, or a phosphorylated oligonucleotide analog is optionally bonded to the 2'-terminal through an alkylene linker. Such an analog can preferably be a sugar derivative wherein the sugar portion is modified; a thioate derivative wherein the phosphodiester bonding portion is thioated; a phosphoryl derivative wherein the phosphoric acid portion at the terminal is substituted; or a purine derivative wherein the purine base is substituted; and is more preferably a phosphoryl derivative wherein the phosphoric acid portion at the terminal is substituted, a sugar derivative wherein the sugar portion is modified, or a thioate derivative wherein the phosphodiester bonding portion is thioated.
The "5'-phosphorylated oligonucleotide analog which has one hydroxyl group removed from the 5'-phosphoric acid group" means a non-natural type derivative of "oligonucleotide" in which 2 to 50 "nucleosides" being the same or different, are bonded by phosphodiester bond linkages, and means a derivative having the following residual group: 0
II
R
(wherein R 6 has the same meaning as defined above) instead of the hydroxyl group at the 5' end of the oligonucleotide.
Such an analog can preferably be a sugar derivative wherein the sugar portion is modified; a thioate derivative wherein the phosphodiester bonding portion is thioated; an ester wherein the phosphoric acid portion at the terminal is esterified; or an amide wherein the amino group on the purine base is amidated; and is more preferably a sugar derivative wherein the sugar portion is modified, or a thioate derivative wherein the phosphodiester bonding portion is thioated.
"Salt thereof" means a salt of the compound of the S :/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification/03.05.05
I
S- 22 present invention, since the compound can be converted to a salt.
Such a salt can preferably be a metal salt such as an alkali metal salt, a sodium salt, a potassium salt and a lithium salt; an alkaline earth metal salt, a calcium salt and a magnesium salt; an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt or a cobalt salt; an amine salt such as inorganic salt, an ammonium salt; or an organic salt, a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N-methylglucamine salt, a guanidine salt, a diethylamine salt, a triethylamine salt, a dicyclohexylamine salt, an N,N'-dibenzylethylenediamine salt, a chloroprocaine salt, a procaine salt, a diethanolamine salt, an N-benzylphenethylamine salt, a piperazine salt, a tetramethylammonium salt and a tris(hydroxymethyl)aminomethane salt; an inorganic acid salt such as a hydrogen halide salt, hydrofluoride, hydrochloride, hydrobromide and hydroiodide; nitrate, perchlorate, sulfate or phosphate; or an organic acid salt such as a lower alkanesulfonate, e.g., methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; an arylsulfonate, benzenesulfonate and p-toluenesulfonate; acetate, malate, fumarate, succinate, citrate, tartrate, oxalate or maleate; or an amino acid salt such as a glycine salt, a lysine salt, an arginine salt, an ornithine salt, a glutamate, or an aspartate.
A "pharmacologically acceptable salt thereof" means a salt of the 2-5A analog of the present invention, since it can be converted into a salt. Such a salt can preferably be a metal salt such as an alkali metal salt, a sodium salt, a potassium salt and a lithium salt; an alkaline earth metal salt, a calcium salt and a magnesium salt; an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt or a cobalt salt; an amine salt such as an inorganic salt, an ammonium salt; or an organic salt; a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N- S:/ChemicalSankyo/FP200343/FP200343s.doc p 893 46/FP-200343/gds-mg/specificaton//03.05.05 0 23 iethylgiucamine salt, a guanidine salt, a diethylamine salt, a triethylamine salt, a dicyclohexylamine salt, an N,N'dibenzylethylenediamine salt, a chloroprocaine salt, a procaine salt, a diethanolamine salt, an N-benzylphenethylamine salt, a piperazine salt, a tetramethylammonium, salt and a tris(hydroxymethyl)aminomethane salt; an inorganic acid salt such as a hydrogen halide salt, hydrofluoride, hydrochloride, hydrobromide and hydroiodide; nitrate, perchlorate, sulfate or phosphate; or an organic acid salt such as a lower alkanesulfonate, methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; an arylsulfonate, benzenesulfonate and p-toluenesulfonate; acetate, malate, fumarate, succinate, citrate, tartrate, oxalate or maleate; or an amino acid salt such as a glycine salt, a lysine salt, an arginine salt, an ornithine salt, a glutamate, or an aspartate.
Specific compounds included in the compound of the above formula of the present invention are illustrated in Table 1.
However, the compounds of the present invention are not limited to these.
(Table 1) 0 0 000 1 111 1 4 1 3 0111 7 8 R -P-E P-E -P-E 3 P-E) P-R R 2 13 14 15 16 R R R R R m fn Exemplif jed Compound No. El E 2 E 3 E 4
RI
1 K 2 K' K' O 2 OC 2
HOH
3 K 3
OCHOH
4 KI 1 K-1 K1 OC 2
H
4 0H
K
1 1 K' 1
K
1 1
OCH
4 0H 6 K 1 1 K 1 1
K'-
1
OC
2
HOH
7 K 1 1 K 1 1 K 1 1
K
1 1 OcHOH 8 K 1 K_1 K''1 OH S-;/Chemical/Sakyo/FP20343/FP2oo343s.doc R 4 R' R' R R' m OH H 0 OH H 0 OH H 0 5H H 0 5H H 0 SH SH H 2.
3H SH H 1 5H H 0 P89346/FP-200343/gds-mg/spccifir-ation//03.05.05 24
K
2 2
K
1 '1 K 3 1 OH
K
2 3
K
1 71 K 3 1 OH
K
2 4
K
11
K
3
OH
K
2 1 K' 2 k 3 1 OH
K
2 -1 1 1' K 3-2 OH
K
2 '1 K 1
K
3 3
OH
K
2 1 K3- 4
OH
K
2
K
3
OH
K2- 2 K'1- 2 K 3 2
OH
K2- 2 K3-' O 2
HOH
K2- 3
K
3 1 O 2
H
4 0H k 2 4 K''I K> 3-1 OC 2
H
4
OH
K'1- 2
K
3
C
2
HOH
K 2- K'1- 1 K 3 2
C)C
2
H
4
OH
K
2 K3- 3
OC
2
H
4
OH
K''l K>- 4
OC
2
H
4
OH
K
2 1 K> 3-5 OC 2
H
4
OH
K2- 2
K'-
2
K
3 2
OC
2
H
4
OH
K1-1 K'1- 2
OC
2
H
4
OH
K'1 1 K'1- 2
OC
2
H
4
OH
K'1 1 K'1-1 K'1- 2
OC
2
H
4
OH
K'1 1 K'1-1 K'1- 2 K'1 1
OC
2
H
4
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
SH H SH H
H
0O(CH2) 3 0H 0 H 0O(CH 2 3 0H 0 H 0O(CH2) 3 0H 0 H 0O(CH 2 3 0H 0 H 0O(CH 2 2 30H 0 H SH 0O(CH 2 2 30H 0 H 0O(CH 2 2 30H 0 H 0O(CH 2 3 0H 0 H 0O(CH 2 3 0H 0 H 0O(CH2) 3 0H 0 H 0O(CH 2 3 0H 0 H 0O(CH 2 4 0H 0 H 0O(CH 2 4 0H 0 H 0O(CH 2 4 0H 0 H 0O(CH 2 4 0H 0 H 0O(CH 2 4 0H 0 H SH 0O(CH 2 4 0H 0 H 0(C8 2 4 0H 0 H 0 0 0 0 0do 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 1 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 1 0 1
K'
1 I K1' 2 K2-1 K3-l K2-1 K3-1 Kl- K3-1
K'
1 i K'i K''i K''i K'i K 1 'i K''1 K' 1 i K 1 KI-1 K''i K' 1 Ki-' K''I K'I K''i K2-1 11 K3-2 KI- Ki K1- K''I K 1 K1' 2 K2-1 Kl' K3-1
K
2 1
KI
1 k3-1 K2- Kl K3- Kl-l K"'l K> 1 i Ki- KI KI-
OH
OH
O
2
H
4 0H
OC
2
H
4
OH
OC
2
H
4
OH
OC
2
H
4
OH
OC
2
H
4
OH
OC
2
HOH
OH
OH
OC
2
H
4
OH
OC
2
H
4
OH
OH
OC
2
H
4
OH
OC
2
H
4
OH
O
2
H
4 0H
OC
2
HOH
48 K>'1 Kl' O 2
HOH
49 K>1' K 1' OC 2
H
4
OH
S:/ChernicallSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mgspecificatioil/03.05.05 0 25 K'1 1 K 1 1
K
1 '1
K
2 -1 K' 1 1 K3- 2 K K 1 K1-2
K
1 1 K1-' K1-2 2-1 Kl-l K 31 *2-1 K1-l K3-1
K
2 1
K
1 -l K3-1
K
2 -1 K1-
K
3 1 -1
K
1 K1-' K'-i K'-l K 1 1 K1-1 K 1 K K1-1 K -1 K2-1 K1-1 K3-2
K
2 K1-
K
3 K2-1 K1-1 K3-1 K2- 1 1-1 K3-1
K
2 1 K1-1 K 3 -1
K
2 e 1
K
3
K
2
K
1 -l K 3 1
K
2 K1-1
K'-I
K 2
K
3 K2' K1-' K3-1
K
2 -1 K' K3-1
K
2 1
K
1 1
K
3 -1_
K
2 K'1 1
K
3 -1
K
2 K'-l K 3
K
2 1 K1- K 3 -1 K 2-1 Kl-l K 3-1
K
2 -1 K-l K 3 K2- K''l K3-'
K
2 -1 K'-l K 3 1
K
2 K1-1
K
3 -1
K
2 K'-l
K
3 -1
K
2 1 3-1 K2-1 K'- 1 3-1 K2-1 K 1
K
3 1 K K 3 1 OH SH SH SH OH SH OH OH
OC
2 HOH OH SH SH OC2H,OH SH SH SH OH SH OH OH
OC
2
H
4 OH OH OH OH
OC
2
H
4 OH SH OH OH
OC
2
H
4 OH OH OH OH OCp 2 HOH SH SH SH
OC
2 HOH OH SH SH OC2H 4 OH SH OH OH OH SH SH SH OH SH OH OH OC2H 4 OH OH SH SH
OC
2
H
4 OH SH SH SH
O(CH
2 3 0H OH OH OH
O(CH
2 40H OH OH OH
O(CH
2 6 0H OH OH OH O(CH2)OH OH OH OH
O(CH
2 3
NH
2 OH OH OH
O(CH
2 6
NH
2 OH OH OH OPh OH OH OH OBn OH OH OH OMe OH OH OH OEt OH OH OH OPr OH OH OH Gly OH OH OH Me OH OH OH Et OH OH OH
CH
2 OH OH OH OH
C
2
H
4 OH OH OH OH Ph OH OH OH
CH
2 Ph OH OH OH
NH
2 OH OH OH NHPh OH OH OH N(Me) 2 OH OH OH N(Et) 2 OH OH OH SMe OH OH OH SEt OH OH OH SPh OH OH OH
O(CH
2 3 0H SH OH OH
O(CH
2 4 0H 0 O(CH2)40H 0 0(CH2) 40H 0 0(CH2) 40H 0 O(CH2)20H 0
O(CH
2 2 0H 0 0(CH 2 2 0H 0
O(CH
2 2 0H 0
O(CH
2 2 0H 0 SH O(CH2) 2 0H 0 0(CH 2 2 0H 0
O(CH
2 2 0H 0
O(CH
2 2 0H 0
O(CH
2 2 0H 0
O(CH
2 2 0H 0 0 1 0 1 0 1 0 1 0 2 0 2 0 2 0 2 0 2 1 2 0 2 0 2 0 2 0 2 0 2 H 0 0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S :/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification/03.05.05 26- 91 K' K' 1
O(CH
2 4 0H SN OH OH H 0 0 92 K 1 1
O(CN
2 60H SN OH OH H 0 0 93 K' K 1 K O(CH,) 8 OH SN ON OH H 0 0 94 K 2 K-1 K0-1 0(CH 2 3
NH
2 SN ON OH H 0
K
2
K
3 1
O(CH
2 6
NN
2 SN OH ON H 0 0 96 K 2
K
1
''K
3 OPh SN OH OH H 0 97 K 2 1
K
3 OBn SN ON OH N 00 98 K 2 -1 K 3 O~e SN ON OH H 0 0 99 K 2 K1_1 K3-1- Oft SN ON OH 00 100 K 2 -1 K 1 OPr SN ON ON N 0 101 K 2
K
3 Gly SN ON OH H- N 0 102 K 2 K Me SN ON ON H 0 0 103 K2- 1
K
1
K
3 Et SN ON ON 0 00 104 K2-1 K11 K 3-1_ CN 2 ON SN ON OH 00 105 K 2 1
C
2
N
4 OH SN ON ON N 00 106 K 2
K
1 1 K 3 Ph SN ON ON N 00 107 K 2 K1-' K 3
CN
2 Ph SN ON OH N 00 108 K 2
K
3
NH
2 SN OH ON N 00 109 K 2
K'
1
K
3 NHPI SN ON OH N 00 110 K 2 K3- N (Me) 2 SN ON OH N 00 111 K 2 -1 K''1 K 3 N (Et) 2 SN OH OH H 0 0 112 K 1
K
3 SMe SN ON ON 00 113 KZ K 3 SEt SN ON OH N 00 114 K 2 K1-1 K3-1_ SPh SN ON OH O 115 K 2
K'-
1
K
3 '1 O(CN 2 3 O NH 2 OH ON H 0 0 116 K 2 K'-1 K 3 1
O(CH
2 4 OH NH 2 ON OH N 0 117 K2-1 K 1-1 K3-1 O(CN 2 6 0H NH 2 OH ON H 0 0 118 K 2 -1 K- O(CN 2 8 ON NH 2 ON ON H 0 0 119 K2-' K'-1 K 3
O(CN
2 3NH 2
NH
2 ON ON N 0 0 120 K 2 '1 K 1 1K3- 1
O(CN
2 6
NN
2
NH
2 ON ON N 00 121 K2-1 K 1 K3-1- OPh NH 2 ON ON N 00 122 K 2 K11 K 3 OBn NH 2 ON OH N 0 123 K 2 1
K
1 1'K 3 Oe NH 2 ON ON N 00 124 K 2
K
3 OEt NH 2 ON ON N 00 125 K 2
K
1 1 OPr NH 2 ON ON N 00 126 K 2 K'-1 K 3 1 Gly NH 2 OH OH N 0 0 127 K 2 -1 K' 'K 3 -1 Me NH 2 ON ON N 00 128 K2-' K1-1K 3 Et NH 2 OH ON N 0 129 K 2
K
1 _1 K 3
CH
2 O NH 2 ON ON 0 00 130 K 2 -1 K11'K 3 -1 C 2
N
4 ,O NH 2 ON ON 0 00 131 K 2 -1 Kl'' Ph NH 2 ON ON 00 S :/ChemicaUSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 -27 132 K 2
CH
2 Fh NH 2 OH OH H 0 0 133 K 2 1 K'11 NH 2 NH, OH OH H 0 0 134 K 2 1 K11-l NHPh NH 2 OH OH H 0 0 135 K21' K'11 K3 N (Me) 2
NH
2 OH OH H 0 0 136 K 2 1
K
3 1 N (Et) 2
NH
2 OH OH H 0 0 137 K 2 K311~ S~e NH 2 OH OH H 0 0 138 K-'K- 1 SEt NH, OH OH H 0 0 139 K 2 1 SPh NH 2 OH OH H 0 0 140 K''i K ~1K11 0OCCH 2 3 0H OH SH SH H 0 0 141 K''I K'1' 0O(CH 2 4 0OH OH SH SH H 0 0 142 K'- 1 KI1 KIl1 0O(CH2) 6 0H OH SH SH H 0 0 143 K 1 '1 K''l 0O(0H 2 8 0H OH SH SH H 0 0 144 K''i ~11 K 1 1 0O(CH2) 3
NH
2 OH SH SH H 0 0 145 K 1 '1 K'1 1 0O(CH 2 6
NH
2 OH SH SH H 0 0 146 K''I KIK1 -1 OPh OH SH SH H 0 0 147 K'1' K 1 K'1' OBn OH SH SH H 0 0 148 K 11 I- ~1-1 OMe OH SH SH H 0 0 149 K 1 K1 K1 O~t OH SH SH H 0 0 150 K' K11 K OPr OH SH SH H 0 0 151 Kii1-1 1-1 Gly OH SH SH H 0 0 152 K 11 K'1- K 1 1 Me OH SH SH H 0 0 153 Ki11 K'1'1K 1 1 St OH SH SH H 0 0 154 K 11 K 1 '1K'1 1
CH
2 OH OH SH SH H 0 0 155 Kl 11
C
2
H
4 OH OH SH SH H 0 0 156 K'- 1 K''-1K' 1 Ph OH SH SH H 0 0 157 K' K 1 1
K
1
CH
2 Ph OH SH SH H 0 0 158 K' K11 K NH 2 OH SH SH H 0 0 159 K''i K''1K'1- NHPh OH SH SE H H 0 0 160 K' K1 Kl N (Me) 2 OH SH SH H 0 0 161 K' K' K' N (Et) 2 OH SH SH H 0 0 162 K 1 '1 Kll Kl1 S~e OH SE SE H H 0 0 163 K 1 'I K' 1 i SEt OH SE SE H H 0 0 164 K''I S~h OH SE SH H. 0 0 165 K 11 K11 K'11 0O(CH,) 3 OH SH SH SH H 0 0 166 K''i K' 1 i 0O(CH2) 4 0H SH SH SE H H 0 0 167 K 1-1 ~11 K 1 1 0O(CH 2 6 0H SH SH SH H 0 0 168 K 11
K
1 'l K''l 0O(CH 2 8 SOH SH SH SH H 0 0 169 K 11 it' 0(CE 2
NH
2 SE SE SH H 0 0 170 it'I K'K 1-1- 0(CE9 6
NH
2 SH SE SH H 0 0 171 K 1- K'l 02h SE SE SE H H 0 0 172 K'1' K'Kl' 1 0Th SH SH SH H 0 0 S:/Chemical/Sankyo/FP200343/FP200343s.doc S:/CerncaL'anlyo/P20043/P20043sdocP89346/FP-200343/gds-mg/specification//03.05.05 28 173 K 1 'I OMe SE SE SE H H 0 0 174 K''1 K''i CEt SH SE SE H H 0 0 175 K''1 K''I OPr SE SE SE H H 0 0 176 K''1 K 11
K
1 'i Gly SE SE SE H H 0 0 177 K' 1 I K'- 1 Me SE SE SE H H 0 0 178 Et SE SE SE H H 0 0 179 K' 1 i K 1
CH
2 0H SE SE SE H H 0 0 180 K' 1 i K' 1 iK''1 C 2
E
4 OE SE SE SE H H 0 0 181 K'- 1
K'-
1 Ph SE SE SE H H 0 0 182 K''1 K' 1 i K' 1
CE
2 Ph SE SE SE H H 0 0 183 K''i K' 1 iK'' NE 2 SE SE SE H H 0 0 184 K' 1
K
1 -I K''I NEPh SE SE SE H H 0 0 185 K''i K'i N (Me) 2 SE SE SE H H 0 0 186 K''1 K''I K 1 N (Et) 2 SE SE SE H H 0 0 187 K''1 K''IK' 1 S~e SE SE SE H H 0 0 188 K''1 K''IK'- 1 SEt SE SE SE H H 0 0 189 K''I K''i SPh SE SE SE H H 0 0 190 K''I K''i K' 1 0(CE 2 3 0H NE 2 SE SE H H 0 0 1,91 K' 1 I K''I 0(CE 2 4 0H NE 2 SE SE H H 0 0 192 K''I K''l K' 0(CE 2 6 0E NE 2 SE SE H H 0 0 193 K 1 'i K''i 0(CE 2 8 0H NE 2 SE SE H H 0 0 194 K''I K''i 0(CE 2 3NE 2
NE
2 SE SE H H 0 0 195 K''1 0(CE 2 6
NE
2
NE
2 SE SE H H 0 0 196 It' it' it' OPh NE 2 SE SE H H 0 0 197 K''i K''i Q~n NE 2 SE SE H H 0 0 198 K''I K''1K 1 1 I OMe NH 2 SE SE H H 0 0 199 it'' Klt' GEt NH 2 SE SE H H 0 0 200 K''I K''IK''1 OPr NE 2 SH SE H H 0 0 201 K''i K''1K''i Gly NE 2 SE SE H H 0 0 202 K''i K''iK''1 Me NH, SE SE H H 0 0 203 K 1 K''i Et NH 2 SE SE H H 0 0 204 K''I K 1 K''l CH 2 0H NE 2 SE SE H H 0 0 205 K''IKII 1
C
2
E
4 OE NE 2 SE SE H H 0 0 206 K''I K''I Ph NE 2 SE SE H H 0 0 207 CE 2 Ph NE 2 SE SE H H 0 0 208 K''I NH 2
NE
2 SE SE H H 0 0 209 K''lK''l NHPh NE 2 SE SE H H 0 0 210 K''l N (Me) 2
NE
2 SE SE H H 0 0 211 K- K''iK''i N (Et) 2
NE
2 SE SE H 0 0 212 K''I K''I S~e NE 2 SE SE H H 0 0 213 K''I K''1 SEt NE 2 SEH SE H H 0 0 S:/ChemicaUSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 29 214 K 1
K
1
K
1 SPh NH 2 SH SH H 0 0 215 K 1
K
1 -2 O(CH2)30H OH SH SH H 0 0 216 K 11
K'-
2 O (CH 2 40H OH SH SH H 0 0 217 K-l K1- 2 O (CH 2 60H OH SH SH H 0 0 218 K' Ki' K 1 2 0 (CH 2 8 OH OH SH SH H 0 0 219 K 1 K K 1 2
O(CH
2 3NH 2 OH SH SH H 0 0 220 K 1 K1-2 O (CH 2 6
NN
2 OH SH SH H 0 0 221 K' 1 K1- K 1 OPh OH SH SH H 0 0 222 K1- 1 K K 1 -2 OBn OH SH SH H 00 223 K1-' K' 1 K1-2 OMe OH SH SH H 0 0 224 K1-' K' K-2 OEt OH SH SH H 0 0 225 K 1 K1- K1-2 OPr OH SH SH H 00 226 K1' K' 1 K1- 2 Gly OH SH SH H 0 0 227 K1- K 1
K
2 Me OH SH SH 0 228 K1-' K' 1
K
1 2 Et OH SH SH H 00 229 K' K 1 1 K1-2 CH 2 OH OH SH SH H 00 230 K1 K1 Kl-2 C 2 H40H OH SH SH H 0 0 231 K' K' 1
K'-
2 Ph OH SH SH 0 232 K1- K' 1 K1- 2
CH
2 Ph OH SH SH H 0 0 233 K1-' K 1 1 K-2 NH 2 OH SH SH H 0 0 234 K' 1 K-2 NHPh OH SH SH H 0 0 235 K' 1 K1- K 1 -2 N (Me) 2 OH SH SH H 0 0 236 K' K 1
K
1 -2 N(Et) 2 OH SH SH 0 237 K 1 K K 1 2 SMe OH SH SH H 0 0 238 K1- 1
K
1 1 K1- 2 SEt OH SH SH H 00 239 K' 2 SPh OH SH SH H 00 240 K' 1 K'-2 O(CH 2 )30H SH SH SH H 0 0 241 K'-1 K' K'- 2
O(CH
2 )40H SH SH SH H 0 0 242 Kl-' 1 K' K 1 -2 O (CH2) 6 0H SH SH SH H 0 0 243 K' K' K1- 2
O(CH
2 )80H SH SH SH H 6 0 244 K1- 2
O(CH
2 3
N
2 SH SH SH 0 0 245 K- K 1 1 K-2 O (CH 2 6
N
2 SH SH SH H 0 0 246 K' K' 1 K 1 -2 OPh SH SH SH H .0 0 247 K1-' K 1 1 K1- 2 OBn SH SH SH H 0 0 248 K' K'- 2 OMe SH SH SH H 0 0 249 K' K 1
K
1 2 OEt SH SH SH H 00 250 Kl' K'-2 OPr SH SH SH H 0 0 251 K 1 K1- K1-2 Gly SH SH SH H 00 252 K1-' K1- K 1 2 Me SH SH SH H 00 253 K' K1- 2 Et SH SH SH H 0 0 254 K1- 1
K
1 1 K'-2 C20H SH SH SH 0 0 S:/Chemical/SankyolFP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05
S
30 K' 1 K1 K 1 2 K''I K'1 2
K
1 'I K'1 2 K''i K'1 2 K''I K'1 2 K''1 K'1 2 K''i K'1 2 K''i K'1 2 K''i KI 1 K'1 2 K''i K K''i K''I K 1-2 K''1 K'1 2 K''1 K''i K 1-2 1- 1 1 1K 1-2 IC KiC K 1- K''i K'1 2 K''i K'1 2 K''1 K'1 2 K''i Kl-' K'- 2 K''I K'1 2 K''i K'1 2 K''I K'1 2 K''i K1' 2 K''i K'1 2 K''i K'1 2 K''I K'1 2 K''i K'1 2 K''I K'1 2 K''1 K'1 2 1-2 K''i K''1 1-2 K''l K''i 1-2 K''1 K''i 1-2 K''i K''i 1-2 K''1 K'1 2 K2-1 3-1
K
2 '1 K 3-1
K
2 1 K 3-1 K2-1 11-- 3-1 K K''l K 3 1 K''1 K'l K-1 C28408 Ph CH2Ph
NH
2 NEPh N (Me) 2 N (Et) 2 S~e SEt SPh o 30H 0 (C2) 40H o 60H o 808 0 (CR2) 3NH 2 o (CR 2 6 t1R 2 0Ph 0Bn OMe QEt aPr Gly Me Et
CH
2 0H 028408 Ph
CH
2 Ph
NH
2 NEPh N (Me),2 N (Et) 2 SMe SEt SPh
OH
OH
OH
0038408 0038408 002H408 SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SR SM SR SR SR SR SR SR
NH
2 SR SR
NH
2 SR SR NH, SR SR
NH
2 SR SR NH, SR SR
NH
2 SR SR
NH
2 SR SH
NH
2 SR SR
NH
2 SR SM
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR NH, SR SR
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR.
NH
2 SR SR
NH
2 SR SR
NH
2 SR SR NH, SR SR POMS OH OH P0MO OH ORH POMS POMS POMS- POMS OH OH POMO Pomo POMO- POMS POHO P0MG
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S:/Chemical/Sankyo/FP200343/FP200343s.doc S:/CerncalSanko/F20043/P20043sdocP89346/FP-200343/gds-mg/specificatian/03.05.05
S
31 2 -l K3- 2 K3- 2 K3'-2
K'-
K'-
K'-
K'-
K'-
K'-
K'-
K'-
K'
K'-
K'-
K>'
K'-
K>'
K>'
K'-
K K K 3- K 3- K K K 3- OC2HOH
OH
OH
OH
OC
2
H
4
OH
OC
2
H
4
OH
OC
2
H
4
OH
OC
2
H
4
OH
POMO
OH
POMO
OH
POMO
OH
POMO
OH
POMO
OH
OH
OH
OC
2
H
4
OH
OC
2
H
4
OH
O
2
HOH
O
2
HOH
O
2
HOH
OH
OH
OH
OH
OH
OC
2
H
4
OH
OC
2
H
4
OH
OCH
4
OH
OC
2
H
4
OH
OC
2
HOH
OC2,OH
OC
2
H
4
OH
OC2HOH
OH
OH
OH
POMS POMS POMS OH POMO OH POMS POMS POMS OH POMO POMO POMS POMO POMS POMS POMO POMO POMO POMO POMS POMS POMS POMS POMS POMO POMS POMO POMO SH POMO SH POMS SH POMS SH SH POMO SH POMS POMO POMO POMS POMO POMO POMS POMS POMS OH POMS ATEO OH SH ATHO SH OH SH ATEO ATEO ATEO ATEO OH SH ATHO SH OH SH ATEO ATEO ATEO OH ATEO OH OH OH ATHO ATES OH SH ATES SH OH
POMS-
OH-
OH-
POMS-
OH-
POMO POMO POMS POMO POMO POMS POMS POMO POMO SH SH SH SH POMO POMS POMO POMO POMS POMS POMS
OH-
OH-
ATEO ATEO ATEO OH
OH-
ATEO ATEO ATEO
OH-
ATEO ATEO
OH-
OH-
ATES-
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
S:/Chemical/Sankyo/FP2003431M00343s.doc S:/CemicL/SnkyaFP20343FP20343sdocP89346/FP-200343/gds-rngspecificadon/lO3.05.05 0 32 337 K" K" OH 338 K 2 1
K
3 1
OH
339 K 2
K
3 '1 OC 2
H
4
C
340 K 2 1 K''I K OC 2
H
4
C
341 K 2 1 Kl>' K 3- )2, 342 OC,H 4
C
343 K-1 K3- 1 OC2, 4 344 K3-' OC,H 4
C
345 K K>'I K OC,H 4
C
346 K'l OC2H 4
C
347 K>'I K 1' K>'i OH 348 K 1' K>'l OH 349 Kl>' OH 350 K 1' K 1' OH 351 K 1' K 1' K 1' OH 352 K 1' K 1 1 OC2,( 353 K K>'l Kl>' OC2, 4 354 K>'i K 1 -1 K 1- OCH 4 355 K>'l OCH 4 356 K> 1 I K>- 1 Kl>' OC2H 4
C
357 K 1' OCH 4 358 K 1' K 1' OC2H 4 359 K 1' OC2, 4 360 Kl>' OH 361 K>'i K 1' K 1 1
OH
362 K 1' K>1' OH 363 K>'1 K>1' K 1' OH 364 K 1' K 1' K 1' OH 365 K''i K 1 1 K 1' OCH 4 366 KI- 1 K>1' OCH 4 367 OCAH 368 OC2H 4 369 K 1' OCAH 370 K>1' K 1' K 1' OCAH 371 K>1' OC 2
H
4 372 K>'l K 1' OCH 4 373 K>1 K'l K>1 OH 374 K-1 OH 375 K 2 1 K>'l K OH 376 K' 1 K OH 377 OH S:/ChemicaIISankyo/FP200343/FP200343s.doC
H
H
H
H
H
H
H
H
H
)H
)H
)H
)H
)H
)H
)H
OH
OH
OH
OH
OH
OH
OH
OH
SH ATES ATES- H 0 0 ATES ATES ATES- H 0 0 ATES OH OH -H 0 0 SH ATES OH -H 0 0 SH OH ATES- H 0 0 SH ATES ATES -H 0 0 ATES ATES ATES- H 0 0 OH ATES OMH H 0 0 OH OH ATES- H 0 0 OH ATES ATES- H 0 0 ATEO SH SH -H 0 0 SH ATEO SH -H 0 0 SM SM ATMO- H 0 0 SH ATMO ATEO- H 0 0 ATEO ATEO ATEO- H 0 0 ATEO SH SH -H 0 0 SH ATEO SH -H 0 0 SH SH ATEO- H 0 0 SH ATEO ATEO- H 0 0 ATEO ATEO ATEO- H 0 0 OH ATEO SH -H 0 0 OH SM ATEO- H 0 0 OH ATEO ATEO- H 0 0 ATES SH SH -H 0 0 SH ATES SM H H 0 0 SH SH ATES -H 0 0 SM ATES ATES- H 0 0 ATES ATES ATES- H 0 0 ATES SH SH -H 0 0 SM ATES SH H 0 0 SH SM ATES -H 0 0 SM ATES ATES- H 0 0 ATES ATES ATES- H 0 0 OH ATES SH -H 0 0 OH SH ATES- H 0 0 OH ATES ATES- H 0 0 PTEO OH OH -H 0 0 SM FTEO OH -H 0 0 SH OH FTEO- H 0 0 SH PTEO PTEO- H 0 0 PTEOPTEO PTEO- H 0 0 P89346/FP-200343/gds-ig/specificadion//03.05.05 0 33
K
2 1 K''l K 3 1
K
2 '1 K''l K 3 1 K Kl-l K 3 1 K-1 K 3 1
K
2 '1 K 3 1 K2-1 11l 3-1 K Kl- K 3-
K
2 1 K''l K 3 1
K
2 1 K''l K 3 -1
K
2 1 K'1-1 K 3 '-1
K
2 1 K'1- 1
K
3 '1
K
2 '1 K 3 1
K
2 1 K 3 1 K2-1 K11 K3-1 K Kl- K3- *2-1 11lK K K- K 3<-1 2 1 Kl-' K 3 '1
K
2 1 Kl-' K 3 '1 k 2 -1 K'-l K 3 1
K
2 1 K 3 1
K
2
K
3 -1 K' K'1 1 K K''I K'1 K''-I *K1- K'I K''-l *K1' K''-1 K11K11K1-1 K 1- K''l K K''l K''-l K''l K''-l K''I K'l K''-l K'l K''-l K''l K K''l K'l K''-l K
O
2
HOH
OC2H 4
OH
OC2HOH
O
2
HOH
OC2HOH OC2,OH OC2H 4
OH
O
2
HOH
OH
ON
OH
OH
OH
O
2
HOH
QC
2
H
4
ON
OC2,OH
O
2
H
4
OH
OC2,OH
O
2
HOH
O
2
HOH
OC
2
N
4
OH
OH
OH
OH
OH
OH
OC
2
H
4
OH
OC
2
N
4
ON
OC
2
H
4
OH
00 2
H
4 0H
OC
2
H
4
OH
OC
2
N
4
ON
OC
2
N
4
OH
OC
2
H
4
OH
OH
OH
OH
OH
OH
OC2H 4
OH
O
2
HOH
PTEO OH OH SN PTEO OH SH OH PTEO SH PTEO FTEO PTEO PTEO PTEO OH PTEO OH OH OH PTEO OH FTEO PTEO FTES OH OH SN PTES OH SH OH PTES SN PTES PTES PTES PTES PTES PTES OH OH SN FTES OH SH OH PTES SN PTES PTES PTES PTES FTES ON PTES OH OH ON PTES ON FTES PTES PTEO SN SN SN PTEO SN SN SH PTEO SN PTEO FTEO PTEO PTEO ?TEO PTEO SN SN SN PTEO SN SN SN PTEO SN PTEO PTEO FTEO PTEO PTEO ON FTEO SN ON SN PTEO ON PTEO PTEO PTES SN SN SN FTES SN SN SN PTES SN PTSS FEES PTES PTES PTES PTES SN SN SN PTES SN
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S:/ChemicaUSankyo/FP200343/FP200343s.doc S:/Cemiallanko/F20033IF20043sdocP89346/FP-200343/gds-mg/specificadion//03.05.05 34 419 K1 420 K 1 421 422 423 424 425 K2-1 K11 K3-1- 426 K2-1 Ki' K 3-1 427 K 2 K''I K 3 -1 428 K2-1 K3-1_ 429 K2-' K''l K3-1 430 K2-' K11 K3-1 431 K2-1 K''l K3-1_ 432 K2-1 K''I K3'-1 433 K2' K''I K3-1_ 434 K2-1 KI- K3-1 435 K2-1 K1 31 436 K2-' K11 K3-1_ 437 K3-' OC2H40 0C2H40 OC2H, 4 0H OC2H,OH OC2H,OH OC2HOH OPh OPh OPh OPh
OH
OH.
OH
OC2HOH OC2H,OH
OC
2
H
4 O8 OC2HOH OC2H,OH OC2H,OH SH SH PTES SH PTES PTES FTES PTES FTES OH PTES SH OH SH PTES OH PTES PTES AIM OH OH ALM AlM OH AU OH ALM AIM AIM ALM SH AULM AUM SH OH ALM SH AIM OH OH AIM ALM OH OH ALM OH AIM OH SH AIM ALM
H
8
H
H
H
8
H
H
H
H
H
H
H
H
H
H
H
H
H
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SH OH ALM! SH AUM OH 438 K' 1 OPh 439 K'1- K1-' OPh 440 K1' K1' K1' OPh 441 K1' K1' OPh 442 K11 OH 443 K11 K1' OH 444 K1' K1' K1' OH 445 K1' K1' K1' OC2HOH 446 K1' K1' OC2H,OH 447 K11 K1' K11 OC2H408 448 K1' K1' K11 OC2H40 449 K11 K' 4
K'
4 OC2H,OH 450 K' K1 K1' OC2H40 451 K2-1 K>'1 K3-1_ OC2HOH 452 K1' K1' OC28408 453 K1' K1' K11 OC2H40 454 K K K'-l OC2H40 455 K1' K1' K1' OH 456 K2_' Kl>' C2840 457 K1' OC2H40 458 K 1K K1' OC2H40 459 K K' K' K1_' C2H408 S:/ChemicaUSankyo/FP200343/FP200343s.doc AUM SH ALM ALM AU'! SH AIM ALM SH ALM SH SH SH AI! OH AIM OH SH OH AI' SH ALM SH SH SH ALM OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH H 00 H 8 0 H 0 0 H 0 0 H 0 0 H 0 0 H 0 0 H 0 0 H 0 0 8 0 H 0 0 H 0 0 H 00 0 2 0 2 0 2 1 2 Li 0 2 L2 ON'' 0 1 12 ON'' 0 1 12 0 1 12 ON1-' 1 1
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH P89346/FP-200343/gds-mgspecification//03.05.05
I~
S
K1 K 1 1
K
1 1 OH
K
2 K1- K 3 1_ OC2
H
OH
K1 K 1 1
K
1 1 OC 2
H,OH
K
1 1 K1 1 K'1 OC 2
HOH
K
1 -1 K' 1
K
1
K
1
OC
2
N
4
OH
K
1 1 K 1 7 1 K OH
K
2 K'1' K 3
OC
2
N
4
OH
K1-' K1-' K1-' OC 2
H,OH
KI K1 1 K 1 '1 OC 2
H,OH
K
1 1
K
1 1
K
1
OC
2
H,OH
K
1 '1 K 1 1 K 1 '1 OH
K
2 1
K'
1
K
3 1
OC
2
N
4
,O
K1 K 1 1 K'1 OC 2
H,OH
K1 K1 1 K 1 1
OC
2
H,OH
K
1 1
K'
1 1 K1- K1-' OC 2
H,OH
K 1 1 K1 1 K' OH
K
2 K1_1 K 3 -1 OC 2
HOH
K 1 1 K 1 1 K 1
OCH
4
OH
Ki K 1 1 K'I OC 2
H
4
OH
K 1 1 K1- K 1
OC
2
H,OH
K
1 1 K 1 1 K1 1
OH
Kl-1 K1-1 K3-1 OC 2
HOH
K'
1
K'
1 i K'I OC,H,OH *11 K'1 1 K1 1
OC
2
H
4
OH
K'-1 K''1 K1'1 K 1
OC
2
H
4
ON
K1 1 K'1 K1 OH
K
2 K3_' OC 2
N
4
OH
K'1 K'1 OC 2
H
4
ON
K1 K'1 1
K-
1
OC
2
H
4
ON
K1-' K 1 '1 K' 1 1 K'1 OC 2
N
4
ON
K
1 1 K1 1
K
1 1
OH
K
2 1 K 3
OC
2
HOH
K'1 1
K
1 1 K 1 1 OC 2
N
4
OH
K1 K 1 1 K 1 1 OC 2
HOH
K-1 K' 1 1 K'1 K'-l OC 2
HOH
K
1 K1 K'1 OH
K
2
K
1 1 K1 OC 2
H
4
ON
K'1 K1 K1 OC 2
H,OH
K
1 1 K''1 K1 1
OC
2
H
4
OH
K
1
K
1 1
OC
2
H
4
OH
K1 K 1 1 K1 OH 35 SH SN OH OH OH SN SH SH OH SN SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SN SH SN OH SN SH SN OH OH OH SN SH SN OH SN SH SN OH ON OH SN SH SN OH SN SH SN OH OH OH SN SH SN OH SN SH SN OH OH OH SN SH SN OH SN SH SN OH ON OH SN SH SN OH SN SH SN
OH
OH
OH
OH
SH ON
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH ON
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH ON
OH
OH
OH
OH
SH OH
OH
L2 ON1 01 L1 ONI-2 0 1
L
1
ON'-
2 0 2 L' ON'1 2 0 2 L1 ON1-212 L ON1-2 0 2 L2 ON' 2 1 2 L2 ON'- 2 0 2 L2 ON1-2 01 L2 O-2 0 1
L
2 0N 12 0 1 1 ON'- 0 L1 ON1-3 0 1 L1 ON' 1- 1 L1 ON 0 1
L
1
ON'-
3 0 2 2 ON -3 o 1 L2 ON 1 3 0 2 L 2 ON1-3 1 L2 ON'- 3 0 2
L
1 ON' 0 2 L' ON 1 -4 0 2 Ll ON 1-4 0 2 2 ON1-4 13 L1 ON1- 0 1 L2 ON1- o L2 ON 0 1 L2 ON1- 2 ON 1-4 o LI 0 1 Ll 0 2 L I ON 1-5 0 2 LI 1 2 ON1-5 2 L2 0 1 L2 0 2 L2 0 2 L2 ON 1 -5 0 L2 ON'_ 0 2
L
2
ON'
4 5 0 1 S :/Chemical/Sankyo/FP200343/FP200343s .dbc P89346/FP-200343/gds-mgspecificasion/03.05.05 0 501 K 3
OCHOH
502 K 1 'i K'I K 1 1
OC
2
H
4
OH
503 KI 1
K'-
1
O
2
N
4
OH
504 K''1 K 1 1 i Kl 1 1
OC
2
H
4
OH
505 K''i K'I KI 1
OH
506 K 2 1 Kl- 1
K
3 1
OC
2
N
4
ON
507 K 11 i K'1 1
OC
2
N
4
ON
508 K 1 1
K
1 1 K'1- OC 2
H
4
ON
509 K 11 i K''i K' 1 I K 1
OC
2
N
4
ON
510 K''i K 1 1
OH
511 K 2 1
OC
2
H
4
ON
512 K''I OC 2
H
4
OH
513 K 1 K'1 1
OC
2
N
4
ON
514 K''1 Kih 1
K
1 K'1 1
OC
2
H
4
ON
515 K''1 K''I OH 516 K 2 1 K 1 1 I K 3 1 OC 2
H
4
ON
517 K 1
KI
1 K'1 1
OC
2
HOH
518 K 1 'i K' 1
K
1 1 i OC 2
H
4
ON
519 K''I K''l K'l K 11
O)C
2
N
4
OH
520 K 11
K'
1 i K''i OH 521 K 2 '1 OC 2
H
4
ON
522 KI 1 K 1 1
OC
2
H
4
OH
523 K'- 1 K''1 K''1 OC 2
H
4
ON
524 K 1 'i K' 1 i K 1 1
OC
2 4
OH
525 K 11 i K 1 1 i K 1 1
OH
526 K 2 1 K3 OC 2
H
4
ON
527 K''I O2N4ON 528 K'-i K 1 K 1 1
OC
2
HOH
529 K 1 -I K'-I K 1
K
1 '1 OC 2
N
4
ON
530 K' 1 i K''i K' 1
OH
531 K2- KiC KC 3- OC 2
H
4
ON
532 K 1 'I K'l OC 2
N
4
ON
533 Kl 1 1
K
1 1
K
1 '1 O)C 2
HON
534 K 1 1 i K' 1 K''i K 1 '1 OC 2
H
4
OH
535 K 1
K
1 1 i K' 1
OH
536 K 2 1
K
1 1 I K 3 '1 OC 2
H
4
OH
537 K'I K''1 K 1 1
OC
2
H
4
ON
538 K 1 'I K'- 1
OC
2
N
4
OH
539 K''l K' 1 I OC 2
N
4
OH
540 KiC KI KI OH 541 K K''I K 3 1 OC 2
H
4
ON
S:/ChemicallSankya/FP200343/FP200343s.doc 36 OH OH OH SN SH SN OH SN SH SH OH ON OH SN SH SH OH SN SN SH OH ON OH SN SH SN OH SN SN SN OH ON OH SN SN SN OH SN SN SN ON ON ON SN SN SN ON SN SN SN OH ON ON SN SN SN ON SN SN SN OH ON OH SN SN SN ON SN SN SN ON ON ON SN SN SN ON SN SN SN OH ON
ON
ON
ON
SN ON
ON
ON
OH
ON
SN ON
ON
ON
OH
OH
SN ON
OH
ON
OH
ON
SN ON
ON
ON
OH
OH
SN OH
OH
OH
ON
OH
SN ON
OH
ON
ON
ON
SN ON
ON
ON
OH
OH
SN OH
ON
OH
L
1 I ON 2 1 0 2 L 0 N2- 0 2 L 10N2- 0 2 L 1 0N 2 '1 1 2 L 1 0N 2 '1 0 2 L2 ON2-1o1 L 2 0N 210 1 L 2 0N 210 1 L2 N2-111 L 2 0N2- 0 1 L 1OIC- 1 1 L 10N 220 1 L1 ON2-212 L I0N 220 2 L'2 0N 2 2 0 2 1 N2210 L2 ON2U 0 2 1 O 2-211 L2 OIU2 12 L1 ON2230 L 1ON2V 0 2 L 1 0N2- 0 1 L2 N2210 L 2 ON 2- 0 1 L2 ON223o L 2 ON2V 0 1 L2 N2230 Li ONU 1 1 L 1OIU- 0 1 L 10N 240 2 LI 241 23 L 1ObU- 0 2 1 N2340 L2 0N2 0 2 12 N2310 L 2 ON 2- 1 2 L2 ON2U 0 2 Ll ON2C 0 1 P89346/FP-200343/gds-mg/specification//03.05.05 0 -37 542 K''i K''I OCR 4 OR OH SR SR OH L' ON>2- 0 2 543 KI KI KI OC 2
R
4 OR SR SR SR OR Ll ON2- 0 2 544 K'l K''l K''l K'l OC- 2 ROR OR SR SR SR OR L' oN2-' 1 2 545 K''I K''i OR SR SR SR OR L' ON> 5 0 2 546 K'lK3-'- OCRHOR OR OR OR OR L 2
ON
2 -5 0 1 547 K''I K''1K''1 OC 2
R
4 OR OR SR SR OR L 2 ON>'5 0 1 548 Ki- KI- KI OC 2
R
4 OR SR SR SR OR L2 0N2 0 1 549 K''I K''1 K''l K''l OC 2
R
4 OR OR SR SR SR OR L 2
QOQ
2 1 1 550 K''IK''1K''1 OR SR SR SR OR L2 ON'5 0 1 551 K-1K''iK3-' OC 2
R
4 OR OR OR OR OR L' ON>1 0 2 552 K' Kl>' Kl>' OC 2
R
4 OR OR SR SR OH L' ON3- 1 0 2 553 OC 2
R
4 OR SR SR SR OR L' ON3-' 0 2 554 K''I Kl' OC2,OR OR SR SR SR OR L' ON 3 1 2 555 K>'i K>'lKl> OR SR SR SR OH L' ON>' 0 2 556 K2-' K 3 OC2ROR OH OR OR OR L2 ON 3 0 1 557 OC 2
R
4 OR OR SR SR OR L 2 ON> 0-11 558 K>'IK>'1K>'1 OC2R 4 OR SR SR SR OR L2 ON>'1 0 1 559 KI ICIC Kll C- OC 2
R
4 OR OR SR SR SR OR L2 Ob- 1 1 560 OR SR SR SR OH L 2 ON> 0- 1 561 K 2 1K>'IK3- OC 2
R
4 OR OR OR OR OH L' ON> 2 0 2 562 OC 2
R
4 OR OR SR SR OR i ON>3 2 0 2 563 00 2
R
4 0R SR SR SR OR L' ON>- 2 0 2 564 IC- IC- KlC IC- OC 2
R
4 OR OR SR SR SR OR Li 0N3 1 2 565 OR SR SR SR OR L' ON> 2 0 2 566 OGROR0 OR OR OR OR L 2
ON>-
2 0 1 567 OC 2
R
4 OR OR SR SR OR L 2
ON>-
2 0 1 568 K>'1 K>'I OC 2
R
4 OR SR SR SR OR L 2
ON
3 2 0 1 569 K'I K>'1 K>'I K' OC 2
R
4 OR OR SR SR SR OR L 2 ON>3 2 1 1 570 OR SR SR SR OR L 2
ON
3 2 0 1 571 K-1- OC 2
R
4 OR OR OR OR OR L' ON>- 3 0 2 572 K>'IK>'IKl>' OC 2
R
4 OR OR SR SR OR L' ON>3 3 0 2 573 K>'IK>'iK>'i OC 2
R
4 OR SR SR SR OR L' ON>3 3 0 2 574 Kl>' 00 2
R
4 0R OR SR SR SR OR L' ON>- 3 1 2 575 K' 4 1 OR SR SR SR OR L' ON> 3 0 2 576 K>'1 K3>1 OC 2
R
4 OR OR OR OR OR L 2
ON>-
3 0 1 577 OC 2
R
4 OR OR SR SR OR L 2
ON>-
3 0 1 578 K>'IK>'i OC 2
R
4 OR SR SR SR OR L 2
ON
3 01 579 K>'i K>'l OC 2
R
4 OR OR SR SR SR OR L 2
ON>-
3 1 1 580 K>'1 K>'iK>'1 OR SR SR SR OH L 2 ON>3 3 0 1 58 211K1 3-1 C2,H OR OR OR OR L1 N3-02 582 K>'iK>'iK>1' OC 2
R
4 OR OR SR SR OR L' ON>- 4 0 2 S:/Chemical/Sankyo/FP200343/FP200343s.doc S:/Cemicl/SnkyoFP20343FP20343sdocP89346/FP-200343/gds-mg/specification//03.05.05 0 -38 583 K''i K OCHOH SH SM SH OH Li ON>3 4 0 2 584 K_1 K_1 K1 OC 2
H
4 OH OH SH SM SM OH L' ON>3 4 1 2 585 K''1 K''i OH SH SH SH OH Li ON3- 4 0 2 58 21 K11 K31 C24H OH OH OH OH L 2
ON-
586 KiC KiC OC 2 HH O H0H S HL N 0- 1 587 K>'1 OC2H 4 OH OH SM SH OH L 2 ONw3 4 0 1 588 K>'1 KIK' OC2H 4 0HOH SH SH S OH L 2 ON 3 4 11 590 IC ICI Ki- OH SH S M SH OH L 2 0N 34o1 590 K1_1 K_1 K1_' OC2H SH SH SH OH i ON> 4 1 012 591 K 2 '1 OC2H 4 OH OH OH OH OH L' ON> 5 1 0 2 592 K>'I K>'iK'-K OCIH 4 0H OH SM SH -H OH L' ON> 5 02 593 IC_ IC IC ,OH SM SH SH OH Li ON 350 2 594 21K- IC- -CI C ,H 4 OH OH SH SH SM OH L2 0N3- 1 595 K>'1 OC2H SH SH SH OH L'2 ON>3 5 021 596 K>i K>'1K OC2H 4 OH OH OH OH OH L2 ON>'1 0 1 59 K1K1_1 K1_1 K1_1 O24H O H S H O 2 N3-511 597 IC_ IC1 IC OC,H OH SH SH OH L2 OIC- 0 1 598 K2-' gKl K31 CCH 4 OH SH SH SH OH Li ON> 5 1 012 599 K''1 K''I K''i K OC,H 4 OH OH SH SH SM OH i ON> 411 600 K''i K''i OCHO SH SH SH OH i oN>'1 012 601 K1_1 3 OC2H 4 OH OH OH OH -H OH L' ON 4 0 2 602 K''I Ki- 1 OC,H OH SH SM OH L' 0N 4 1 0 2 603 K2'1 OC2H 4 OH SH SH SH OH L' 0N 4 '1 021 604 K''i K>'1 K>'i K OC2H 4 OM OH SM SH SH OH L2 ON 4 1' 1 605 K''1 K''I OCH40 SH SM SH OH L' ON 4 -1 0 2 606 K>'i K'-I 1 K' OC2HOH OH OH OH -H OH L2 O4-1 0 1 610 K''1 K''iK' OC,H OH SM SH OH L' 2 0N 4 0 1 608 OC2H 4 OH SH SH SH OH Li ON 4 012 609 K''i K''I K' OC2H 4 OH OH SH SH SH OH Li ON 4 '2 1 1 610 K'1 K''1 K''1 OCH40 SH SH SH OH i O o 4 -2 012 315 OiIK- ii- O H OH OH OH L' N4-202 611 K' K OC 2
H
4 OH OH O0H HL N 21 612 K''i K''I OC2,OH OH SH SH OH L2 ON 4 -2 021 613 K''i OC 2
H
4 OH SM SH SH OH L' 0 N 4 2 021 614 K''1 K''1K''1 K'1 OC2H 4 OH OH SH SM SH OH L' 2 0N 4 2 121 615 K>'i K>'i OH SH SH SM OH L' 0N 4 2 021 621 K>'I K>'1 OC2H 4 OH OH OH OH OH iJ ON 4 -3 0 1 617 K''I K''IK'1' OC2,OH OH SH SH OH L' ON 4 -3 012 623 K''I K 1 1 OC2H 4 0H SM SM SH OH L' 0N 4 3 0 2 S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 0 -39- 624 KiLi Ki Ki-i OC 2
R
4 OR OR SR SR SN OH Li ON- 3 1 2 625 K''1 K''iK''I ON SN SN SR OH L0 0N 4 0 2 626 K 2 Kliiti-' OC 2
H
4 OH OH OH OH OH L2 0N 4 3 0 1 627 KIIK-IK- OC 2
H
4 OH OR SR SR OH L2 ON4 3 0 1 628 K''i OC 2
H
4 OR SR SR SN OH L2 0N 4 3 0 1 629 K'i it'K11 1
OC
2
N
4 ON ON SR SR SN OR L2 0N 4 3 11 630 KIt' KiI Itt OR SR SR SR OR L2 ON4 3 0 1 631 it' 1_ K' 4 i' OC 2
H
4 ON OR OH ON OH LP ON4- 4 0 2 632 K'1 KIti K''I OC2R 4 OR OR SN SN ON L' ON 4 4 0 2 633 K' 4 i K''iK''i OC 2
N
4 OR SR SN SN OH Li O 4 4 02 634 K'I K''1K1'1 K'1 O)C2R 4 O OR SR SR SR ON L' 0N 4 4 1 2 635 K 11 I Ki-Kll1 OR SR SR SR ON Li 0N 44 0 2 636 K 2
'K''K
3 OR0 OR. OR OR OR L 2
ON
4 4 0 1 637 KIt' K''IK''i OC 2
R
4 OR OR SN SR ON L2 ON 4 4 0 1 638 Ki K'1 it' OC 2
N
4 OR SR SN SR OH L2 ON 4 4 0 1 639 K''i K' 4 K1_1 K1' 00 2
R
4 0R OR SR SR SR OR L2 0N 44 1 1 640 Kit' K''1 K1' ON SN SR SR OR L2 ON 44 0 1 641 K 2 1 K OC 2
N
4 OR ON OR ON OR L' 0N 4 1 0 2 642 Kili K''IK''i OC 2
R
4 OR ON SR SR OH L' ON''5 0 2 643 K''1 K1l1K1ii OC 2
R
4 OR SR SR SN OR Ll ON 4 -5 0 2 644 K''1 K''i K''i Ki OC2R 4 OR OR SR SR SR ON Li ON 4 1 2 645 K''i K 1 '1 K''l OR SR SN SR OR L' 0N 4 -5 0 2 646 K 2 1K''IK 3
OC
2
R
4 ON OR ON ON OR L2 ON 4 0 1 647 OC 2
R
4 OR OR SR SR OH L2 0N 4 0 1 648 it' K''iK''i OC 2
R
4 OR SR SN SR OH L2 ON 4 -5 0 1 649 Ki1 K''i K''i OC 2
N
4 OR ON SR SN SR OR L2 0N 4 1 1 650 K''1 OR SN SR SN OH L2 0N 4 0 1 1 K1t' K'T'i' OC2N 4 OR OR OR ORH NL N' 0 652 KilI K''iK''1 OC2R 4 OR OR SR SR ON L' ON5-' 0 2 653 K''I OC 2
R
4 OR SR SN SR ON L' 0N 5 1 0 2 654 K''1 K'1 K''1 OC 2
R
4 OR OR SR SR SR OR L' ON'' 12 655 K''l K 1 OR SN SR SN OR L' ON'' 02 656 K 2 '1 K 3 OC2N 4 ON OR OR OR OR L2 ON 5 0 1 657 KiKiiKll1 OC 2
N
4 OR ON SR SN OR L2 oN5t' 01 658 K''i K'1 it' OC 2
R
4 OR SN SR SN OR L 2 Ow''1 0 1 659 K'i Kii K'1 OC2N 4 OR OR SN SN SR OR L2 ON'' 1 1 660 KiKiKii ON SR SN SR OR L2 ON5' 0 1 661 K2_1 K11 g3i1 OC 2
R
4 ON OR ON OR OR Li 0N 52 0 2 662 K''1 K''I OC 2
R
4 OR OR SR SR OR L' ON''2 0 2 663 Kili K- i-i OC2N 4 ON SN SR SN OR L' ON'- 2 0 2 664 IC_ K1_ K11 1_ OC 2
R
4 OR OR SR SN SR OR Ll 0N5 1 2 S:/ChemicaUSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 665 K' 1 i K''I K 1
OH
666 Kl' K 3
OCH
4
OH
667 K 1 'i K'- 1
K
1 1 i OCH,0H 668 K 1 'I K'- 1
K
1 'I OC 2
H
4
OH
669 K'I K'I K 1 1 i K' 1
OC
2
H
4
OH
670 K 1 'i K''i K 1
OH
671 le- IC- OC 2
H
4
OH
672 K''I K 11
K
1 'I OC 2
H
4
OH
673 K 1 i K 1
K
1 'I OC2H 4
OH
674 K''i K''I K''I OC 2
H
4
OH
675 K''I K''I K 1 1
OH
676 K 2 1 K''1 K 3 1 OC2H 4
OH
677 K''i K 1 1 i K 1 1
OC
2
HOH
678 K''i K' 1 I OC 2
H
4
OH
679 K''I K''I K 1 1 K''I OC 2
H
4
OH
680 K 1 'I K 1 1
K
1 1 i OH 681 K 2 1
OC
2 HOHi 682 K 11 i K 1 1 i Ki(- OC 2
HOH
683 K''I K''I K' 1
OC
2
HOH
684 K' 1 i K''i K' 1
K'
1
OC
2
H
4
OH
685 K''I K''I K 1 1
OH
686 K 2 1 K'- 1
K
3 1 OC 2
H
4
OH
687 K''I K'- 1
*K
1 1
OC
2
H
4
OH
688 K''i K''i OC 2
H
4
OH
689 K' 1
K'
1 K1' 1 K_ OC 2
H
4
OH
690 K''I K''I K 1
OH
691 K 2 '1 K 1 1
OC
2
H
4
OH
692 K 1 'i K 1 1
K
1 1 i OC2HOH 693 K 1 1 i K 1 1 K''i OC 2
H
4
OH
694 K''l K 1
K
1 'l K 1 '1 OC 2
H
4
OH
695 K''I K 1 1 K''I OH 696 K 2 1 K 1 1 k3- oC- 2
H
4
OH
697 KiC KiC KI OC 2
H
4
OH
698 K 1 '1 KIC KIC OC 2
H
4
OH
699 KIC KI KIC KI OC2H 4
OH
700 K''I K'I K'- 1
OH
701 IC_ IC IC_ OC 2
H
4
OH
702 KIC IC KIC OC 2
H
4
OH
703 KI IC IC OC 2
H
4
OH
704 IC KIC KI IC oc- 2
H
4
OH
705 K 1 1 i K' 1
K'
1 i OH S:/Cbemical/Sankyo/FP200343/FP200343s.doc 40 SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SR OH OH OH SH SH SH OH SR SH SH OH OH OH SH SH SR OH SR SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SR OH
OH
OH
OH
OH
SR OH
OH
OH
OH
OH
SR OH
OH
OH
OH
OH
SH OH
OH
L' ON 5 2 0 2 L2 N5-2 1 L 2 0NI- 0 1 L2 N5-2 1 L 2 ONU 0- 1 L2 N5-2 1 L I 0N 530 1 L I 0N 1 1 Li 0N5 0 1 L 1
ON
5 3 0 2 L' 0N 5 3 0 2
L
1 2 0N 5 3 0 2 L2 0N 5 3 o12 L2 ON5-3o1 L 2 ONU 0- 1 L2 ON5-3o1 Li ON 540 1 L 1 0NI- 0 1 L I 0N 1 1 L2 ON534o L 2 ONIC 0 1
L
1 2 0N 5 4 0 2 L' ON 5 -5 0 2 L 1 0NS 5 0 2 L' ON 5 -5 1 2 L' ON 5 4 0 2 L2 ON545o L 2 0N 550 1 L2 ON5450 L 2 ON'- 0 1 L2 N545C Ll 0N6 0 1 Ll ON 61 1 L 1 0N'- 0 1 L 1 0N 5 5 0 2 Ll 0N 5 5 1 0 2 P89346/FP-200343/gds-mglspeciflcation//03.05.05 0 706 Ke' OC,HOH 707 K''1 K''1 OC 2
HOH
708 K''1 K''1 OC 2
HOH
709 K 1
OC
2
H
4
OH
710 K>'i K>I KIt' OH 711 OC2,OH 712 K>'i K>'I 0C 2 H,0H 713 K>'I K>'1 0C 2 H,0H 714 K1_' K1' OC 2
N
4
OH
715 KIt' K1_' OH 716 OC 2
HOH
717 K>'i K 1' OC 2
N
4
ON
718 K>'1 K 1 1
OC
2
N
4
ON
719 K>'i Klt' OC 2
H
4
OH
720 K>'1 K'i it' OH 721 K 2 -1 it' K OC 2
HON
722 K>'i K 1' OC 2
H
4
ON
723 K>'i OC2,H 724 K>'1 OCN 4
OH
725 K1_' K'i KIt' ON 726 Kl>' K' OC 2
N
4
OH
727 K>'I K>'I K 1' OC 2
HOH
728 K>'i K 1' 00 2
N
4 0H 729 K>'I K1' OC 2
H
4
OH
730 it' it' Kit' OH 731 K 2 K_1 OC 2
N
4
OH
732 K>'1 OC2,OH 733 K>'i K 1' OC 2
H
4
OH
734 K>'i OC2,OH 735 K'1 it' K>'I OH 736 K>'1 K> 1
OC
2
HOH
737 K>'i K 1' OC 2
N
4
OH
738 K1>' K1>1 K1' OC 2
N
4
OH
739 K>'i K>'1 K>'i K1' OC 2
H
4
OH
740 K>'1 Klt' OH 741 K 2 K3_' OC 2
HOH
742 K>'I K OC 2
N
4
OH
743 K>'it' KIt'K OC 2
H
4
OH
744 K- KI- K' OC 2
N
4
ON
745 K>'i K1_' OH 746 K 2 '1 K>'1 C 2
HOH
S:/ChernicailSankyo/FP200343/FP200343s.doc 41. OH OH OH SH SH SN OH SN SH SN OH OH OH SH SN SN OH SH SN SN OH OH OH SN SH SH OH SH SH SH OH OH OH SH SH SH OH SN SH SN OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SN SH OH OH OH SN SN SN OH SH SN SH OH OH OH SH SH SN OH SH SH SH OH OH
OH
OH
ON
SH OH
OH
OH
ON
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SN OH
OH
OH
OH
OH
SH OH
OH
OH
OH
ON
SH OH
OH
ON
OH
OH
SN OH
OH
OH
OH
OH
SN OH
ON
OH
L 2 ON 6- 0 1 L12 ON~ 0- 1 L 2 ONE'1 0 1 L ON6_' 1 1 L2 ON6-1o Li ON 620 1 L' O 6-2 0 2 1 O 6-2 0 2 L' ON 6 2 0 2 L' 1 6-2 1 2 L'2 ONF 2 0 2 L2 6-2 1 L 2 ONI- 0 1 LiJ O 6-2 0 1 L 2 0N r20 1 2 I N6-2 L I ON" 0 1 L' O 6-3 0 2 L' ONr 3 0 2 L' O 6-3 0 2 L? 2 N6-3 0 2 L2 6N -3o1 L 2 ONr- 0 1 L2 N6-311 L 2 ON6- 0 1 2 O 6-43 L I ONr- 0 1 L 1 0N" 1 1 L2 ON6-3 L 2 ON 0 1 L'2 O 6-4 0 2 L1 O 6-411 L 1 ON6- 0 2 Ll ONb6- 0 2 L1 N6-4 L 1 ON 651 2 Ll ON 650 1 L 2 ON 0 1 P89346/FP-200343/gds-mg/specification/l03.05.05 42 747 K' 1 I K 1
OC
2 HOH OH SH SN OH L 2 ON" 0 1 748 K''I K 1 1
OC
2 HOH SN SH SN OH L 2 ON6- 0 2.
749 K 11 I K''1 K 1 'i K' OC 2
N
4 OH OH SH SN SH OH L 2 ON 6 5 1 1 750 Ki- K KI 1 OH SN SN SN OH L 2 0N6- 0 1 751 K 2 1
K
1 iK 3
O
2 HON OH OH OH OH Li ON 710 2 752 K 1 1 K 1 1
OC
2
N
4 ON OH SN SN OH Li ON7-1 0 2 753 K 1 1
K
1 1 K 1- OC 2 HOH SN SH SN OH Li ON 7 1 0 2 754 K' 1 i K 1 'I K 1 K 1- OC 2
N
4 OH OH SH SN SH OH Li ON 7 -1 1 2 755 KI- K Ki- OH SH SN SN OH Li ON 710 2 76 K2- K OC 2
N
4 OH OH OH OH OH L 0N7- 02.
757 K'I K 11
K
1 1 OC 2 HOH OH SH SN OH L 2 0N 11 758 K''iK''IK 1
OC
2 HON SH SH SN OH L12 ON 7 1 0 1 79 Kl- Kl- Kl- Kl OC 2 HON OH SH SH SN OH L 2 0N 711 1 760 K 11 i K 11
K
11 I OH SH SN SH OH L12 ON 7 -1 0 1 761 K 2 'Kl 1 1
K
3 1 OC2HOH OH OH OH OH L 1
ON
7 2 0 2 762 K 1 1 iK 1
'IK
1
OC
2
N
4 OH OH SN SN OH L 1
ON
7 2 0 2 763 K'I K 1 K 1 1
OC
2
N
4 OH SH SH SN ON L1 ON 7 2 0 2 764 Kl- Ki- KI K 11OC 2
H
4 ON OH SH SN SH OH i ON 721 2 765 K 1
'IK
1 1 iK 1 1 I- OH SN SH SN OH L1 ON '1 2 0 2 766 K 2 1
KI
1
K
3 1 OCHOH ON OH OH OH L 2 ON 7 2 0 1 767 K' 1 i K 1 1 K 1 1
OC
2 HOH OH SH SN OH 1 2 ON 7 2 0 1 768 Ki- Ki- K 11- OC 2 HON SN SN SN ON L 2 0N 720 1 769 K''i K 1
K'
1
K
1 -1 OC 2
N
4 ON ON SN SN SN OH L 2 ON 7 2 1 1 7 70 K'- 1 KlliKP 4 -1 ON SN SN SN OH 1 2 ON 7 2 0 1 771 K 2 OCHOH ON OH OH ON L1 ON 7 3 0 2 772 K 1 1 iK 1 'IK 1 1
OC
2
N
4 OH OH SN SN OH Li ON 7 3 0 2 773 K- K K 1- O 2 HOH SN SN SN ON i ON 730 2 774 K 11 i K 11 Kl 11
K
1
O
2
H
4 OH OH SN SN SN OH i 0N7- 1 2 775 K''l K''IK 1 1 ON SN SN SN ON Li 0N' 7 0 2 776 K'K'K 3 OCHON ON ON ON ON L 2 ON 7 3 0 1 777 Ki- K K1- O 2 HON OH SN SN OH L2 0N7 0 1 778 K 1 1 K-IK 1 1 K- OC 2 HOH SN SN SN ON L12 ON 7 3 0 1 79 KI- Kl- Kl Kl- OC 2
N
4 OH ON SN SN SN OH L 2 0N 731 1 780 K' 1 K'IK''-I ON SN SN SN OH 1 2 ON 7 3 0 1 781 K2- K K3- 1
OC
2
N
4 ON ON ON ON OH Li O -02 782 K 1 1
IK
1 K 1 1
OC
2
H
4 OH ON SN SN ON L1 ON 7 4 0 2 783 K 11 I K 1 l Kl'-l OC 2
H
4 OH SN SN SN ON L1 ON 7 4 0 2 784 KI KiCI KC I- O 2
H
4 ON OH SN SN SN OH i ON 741 2 785 K 11 I K 1
'IK'
1 i ON SN SN SN OH L1 ON 7 -4 0 2 786 IC1 l- I OC 2
N
4 ON ON OH OH ON L 2 0N 0 o1 77 K1-1 K11 K1-1 C24H ON SN SN O ON7-1 N 1 S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 43 788 K''i K''iK''i OC2H 4 OE SE SE SE OH nJ ON7- 4 01 789 K1_1 K> 1 i Kl' OC 2
E
4 OE OH SE SE SE OH L 2 ON7- 4 11 790 K''i K>'1 OH SE SE SE OH L2 ON,- 4 0 1 791 K2-' OC2,OH OH OH OH OH L' ON>- 5 0 2 792 K 1 1 K''1 K 1 OC2,OE OH SE SE OH L' ON>_ 5 0 2 793 K>'I K>'I OC2,OH SE SE SE OH LP ON>- 5 0 2 794 K>'I K>'1 K' OC2E 4 OH OH SE SE SE OH L' ON>' 1 2 795 K>'I K>'I OH SE SE SE OH L' ON>'5 0 2 796 K' OC2,OE OH OH OH OH L 2 ON>'5 0 1 797 K>'i K>'1 OC2,OH OH SE SE OH L 2 0 1 798 K>'I K>'I OC2,OH SE SE SE OH L2 ON>'5 0 1 799 K>'1 K>'1 OC2H 4 OE OH SE SE SE OH LZ ON>'5 1 1 800 K>'i K>'1K>'1 OH SE SE SE OH L2 ON>'1 0 1 801 K2_ OC2,OE OH OH OH OH Li 0N8 0 2 802 K>'i K>'1K>'I OC2E 4 OE OH SE SE OH L' ON8>' 0 2 803 K>'i K>'1K>'I OC2,OE SE SE SE OH L' 0 2 804 K>'i OC2,OE OH SE SE SE OH L' ON>'1 1 2 805 K>'I K1_' OH SE SE SE OH L' 0 2 806 Ke>' OC2H 4 OH OH OH OH OH L2 0 1 807 K>'I K>'i OC2,OH OH SE SE OH L2 0 1 808 K>'i K' OC2,OH SE SE SE OH L2 0 1 809 K>'I K1' K1_1 OC2,H OH SE SE SE OH L2 1 810 K>'I K>'i OH SE SE SE OH L2 ON>'1 0 1 811 K2>' K"1K 3 C)2HOH OH OH OH OH L' ON 8 -2 0 2 812 K>'1 K>'iK>'I OC2E 4 OE OH SE SE OH L' ON>'2 0 2 813 K>'I OC2H 4 OH SE SE SE OH L' ON>'2 0 2 814 K>'I K>'i KI' K' OC2,OH OH SE SE SE OH L' ON8-2 1 2 815 K>'I K>'1K>'i OH SE SE SE OH L' ON>'2 0 2 816 K' K3>' OCEHOE OH OH OH OH L2 ON 8 0 1 817 K>'i Ki- OC2,H OH SE SE OH L2 ON>'2 0 1 818 K>'i K>'i OC2E 4 OE SE SE SE OH L2 ON 8 0 1 819' K>'i K1_' K1_1 K1' OC2H 4 OE OH SE SE SE OH L2 ON>'2 1 1 820 K> 1 I K>'iK>- 1 OH SE SE SE OH L2 ON>- 2 0 1 821 K2>1 K3>' OC2H 4 OH OH OH OH OH L' ON>'3 0 2 822 K>'i K>'1K>'i OC2E 4 OH OH SE SE OH L' ON>'3 0 2 823 K>'i OC2H 4 OH SE SE SE OH L' ON>'3 0 2 824 K>'1 K1_' OC2,OH OH SE SE SE OH L' ON>'3 1 2 825 OH SE SE SE OH L' ON>'3 0 2 826 K>'1 OCE 4 OH OH OH OH OH L2 ON>'3 01 827 K>'1 OC2H 4 OE OH SE SE OH L2 ON 8 0 1 828 OC2H 4 OH SE SE SE OH L2 ON 8 0 1 S:/Chemical/Sankyo/FP200343/FP200343s.doc S:/Cemial/anko/F20033/F20043sdocP89346/FP-200343/gds-mgspecification//03.05.05 0 -44 829 K''I K 11 I K' 1
K
1 1 i OC 2 HOE OH SE SE SE OH L 2 ON- 1 1 830 K' 1 I K''1 K' 1 OH SR SH SH OH L 2 0N 8
I
3 0 1 831 K 2 1 K''1K 3 -1 OC 2 H,OH OH OH OH OH L' ON 8 4 0 2 832 K 1 'I K'- 1
K
1 'i OC 2
E
4 OH OH SE SE OH L 1 0 2 833 K 1 1
K'
1
OC
2 HOE SH SH SE OH i) ON 8 4 0 2 834 K'- 1 K''iK' 1
K'
1
OC
2
H
4 OH OH SH SE SE OH L' ON 8 4 1 2 835 K 1 1 I K''IK' 1 I OH SH SH SE OH i ON 8 4 0 2 836 K 2 Kll OC 2 HOH OH OH OH OH L 2
ON
8 4 0 1 837 K''1 OC 2
H
4 OH OH S SH OH L? 2 0N 8 4 0 1 838 K''I K'I KI 1
OC
2
H
4 OH SE SH SE OH L2 ON B 4 0 1 839 K'_ 1 K'1 K'_ 1 K''1 OC 2
H
4 OE OH SE SE SE OH iJ ON 8 4 1 1 840 K''1 K' 1 i K' 1 OH SE SE SE OH L2 ON 8 4 0 1 841 K 2 '1 K 1 '1 K 3 1
OC
2
E
4 OE OH OH OH OH L' ON 8 5 0 2 842 K 1 1 i K''1 K 1
OC
2
H
4 OH OH SE SE OH L' ON8t 5 0 2 843 K 1 'i K''i OC 2
E
4 OE SE SH SH OH Li O8t' 0 2 844 K' 1 I K''1 K''l OC 2
H
4 OH OH SE SE SE OH i O8t' 1 2 845 K 1 'i K' 1 i K' 1 OH SE SE SE OH L 1 O8-' 0 2 846 It-' K''lK 3
OC
2
H
4 OH OH OH OH OH L2 Ow 9 0 1 847 It' Kit Kit 1
OC
2
H
4 OH OH SH SE OH L2 Ot' 0 1 848 K''i K 1
OC
2
H
4 OH SE SE SE OH L2 ON 8 0 1 849 K''I K''I K' 1 K''I OC 2
H
4 OH OH SE SE SE OH L2 OSt' 1 1 850 Ki- KliKlItl OH SE SH SE OH L2 Ot' ci 851 K 2 '1 K' OC2E 4 OH OH OH OH -OH Li ON9- 0 2 852 K 1 'I K''IK' 1 i- OC2H 4 OH OH SE SE OH L' ON 9 0 2 853 K''1 OC 2
E
4 OH SH SH SH OH L' ON9-' 0 2 854 K''I K''l K 1 1 K''l OC2H 4 OH OH SE SE SR OH L0 ON 9 1 2 855 K''1 K 1 i KI 1 OH SR SE SR OH L 1
ON
9 0 2 856 K'_1K' 1 t- OC 2
H
4 OH OH OH OH OH L2 ON9_' 0 1 857 K''1 K' 1 K'I OC 2
H
4 OE OH SE SH OH L2 ON 9 1 0 1 858 K''1 K 1 'IK''I OC 2
H
4 OE SE SR SE OH L0 ON9_' 0 1 859 K 1 1 I K''I K 1
OC,E
4 OH OH SE SE SE OH L2 N9-' 1 1 860 K''I K''1 OH SE SE SE OH L ON 9 1 0 2.
861 K 2 K3_1 OC 2
H
4 OH OH OH OH OH L 1 ON 9- 2 0 2 862 K 1 1 K''iKI 1
OC
2
H
4 OH OH SE SE OH L 1 O 9-2 0 2 863 K''1 OC 2
E
4 OH SE SE SE OH L 1 ON 9 2 0 2 864 K 1 1
K
1 '1 K''l OC 2
H
4 OH OH SE SE SE OH 0l ON 9- 2 1 2 865 K''i K''i K' 1 OH SR SR SE OH L 1 N9-2 0 2 866 K2_ t- OC 2
E
4 OH OH OH OH OH L2 N9-2 0 1 867 K''1 K' 1
OC
2
H
4 OH OH SE SR OH L2 N9-2 0 1 868 K''I OC 2
H
4 OH SR SE SE OH L2 O 9-2 0 1 869 K1W 1Ki- l OC 2
H
4 OH OH SE SE SR OH L0 ON2 1 1 S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mgtspecifiration//03.05.05 0 45 OCH40 K 11K 11Ki- OC 2
H
4
OH
K K K''I OC 2
H
4
OH
K K OC 2
H
4
OH
K 2-1 K'1 K 3 '-1 K 1- K 1 -1 K 1- K 1- K 1 1 K 1- Kl'' K 1- K 1- K''i K''I K'I K 2-1 K1_1 3-1 K''1 K_1 K1 K'1' K 1- K 1 -1 K 1 K''I K'1 K''-i K2-1 K1-1 K3-1 K''I K 1 K 1- K 1 1
K
11 I K 1 K''I K 1 1
K-I
K2-1 K11 K3-1 K'I K_1 K' 1 K''i K K_1 K_1 K1
K
2 1 K_1 K 3 '-1 K K_1 K' 1 K''I K 1 1
K
2 1 K1_1 K 3 1
K
2 1 K 1 _1 K 3 1
K
2 1 Kl~' K 3 1
K
2 'I K1-1 k 3 2
K
2 1 K 3 2
K
2 1 KI- K 3 2 K_1 K 11K 3 1 K1-1 K11 K3-1_ K K_1 K 3 -1
OH
OC
2
HOH
OC
2
H
4
OH
O
2
HOH
OC
2
H
4
OH
OH
OC
2
HOH
OC
2
HOH
OC
2
H
4
OH
O
2
HOH
OH
OC
2
HOH
O
2
H
4
OH
O
2
H
4
OH
O
2
HOH
OH
OC
2
HOH
O
2
H
4
OH
O
2
H
4
OH
OC
2
H
4
OH
OH
O
2
H
4
OH
OC
2
H
4
OH
OC
2
H
4
OH
OC
2
HOH
OH
O
2
H
4
OH
OH
OC
2
HOH
00 2
HOH
OH
OC
2
HOH
OC
2
HOH
O
2
HOH
OH
OC2HOH SE SH SH OH OH OH OH SH SH SH SE SH OH SH SH SH SH SH OH OH OH OH SH SH SH SH SH OH SH SH SH SH SH OH OH OH OH SH SH SH SH SH OH SH SH SE SH SH OH OH OH OH SH SH SH SH SH OH SE SE SH SH SH OH OH OH OH SH SH SH SH SH OH SH SH SH SH SH OH OH OH OH SH SH SH SH SH OH SH SH SH SH SH OH POMS OH POMS POMS OH POMS POMS OH OH POMS OH POMS POMS OH POMS POMS OH OH POMS OH POMS POMS OH POMS POMS OH OH POMS OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SE OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
L2 ON9-2o1 L 1 ON'- 0 1 L oN9 3 0 2 L I ON 9 3 0 2 L' ON 9 3 0 2 L'1 ON 9 3 0 2 L 2 ON 9 3 0 2 L 2 ON 9 3 0 1 L 2
ON
9 3 0 1 L2 ON"13 L 2 ON'- 0 1 L I ON9_ 1 1 L 1 0N9_ 0 2 L I ON 9 4 0 2 L ON 9 4 0 2 L I ON 9 4 0 2
L
1
ON&
4 012 L'2 ON 9 4 0 2 L2 N9-4 1 L 2 ON" 0 1 L2 ON9-4o1 L 1 ON 950 1 1 ON 9 4 0 1 L I N9-' 1 1 Ll ON_ .0 1 L 2 ON 9 5 0 2 L'2 ON 9 5 0 2 L 2 ON 9 5 0 2 L2 ON9-511 L 2 ON9- 0 1 H H H H H H S:/Chemical/Sankyo/FP200343/FP200343s.docP846F-04/gsmlpcfato/3.55 P89346/FP-200343/gds-mg/specification//03.05.05 46 K1-1 K1-1 K3-2 K'-1 K'1 K3-2 K2-1 K1-l K1-l K2-1 K1- K 1-1 K' K'1 K'
K
2
K
1 -1 K
K
2 -1 K''I K 1-2 K2-1 K1-1 K 1-2 2-1 Kl-l K 3-1 K2-1 K 11 K3-1
K
2 K'-l
K
3 -2 K2-' K1-1
K
3 -2 K2-1 K1-1
K
3 -2 K K K 3 2 -1 K1-1 K1-' K 3- K'-1 K1-' K 3 1 K1-1 K3-2 K1-' K3-2 K11K11K3-2 K2-1 K K 1 1-1
K
2 K K 1 1-1 K' K'- K2-1 K1-1 K 1-2
K
2 -1 K1-1 K 1-2
K
2 K'1' K '1-2
K
2 1
K
1 -l K 3 1 K2-1 K1-1 K 3 -1 Ke'' K1- K 3 -2
K
2 K1-1 K3-2 K2-1 K'-l K 3 -2 K'-l K3-'
K
3 -1
K
3 1- K'-1 K''1 K 3 -2 K 1-1 K -1 K-2
K
3 -2
K'
1
K'
1 K 2-1 K1-1 K 1-1 K2-1 K1- K 1-2 K- K1-1 K1- OC2H 4
OH
OH
OC2H,OH
OH
OC2H 4
OH
OC2H 4
OH
OH
OC2H,OH
OH
OC2H 4
OH
OC2H,OH
OH
OC2HOH OC2H,OH OC2H,OH
OH
OC2H,OH OC2HOH
OH
OC2H,OH
OH
OC2H 4
OH
OC2H 4
OH
OH
OC
2
H,OH
OH
OC2H 4
OH
OC2H 4
OH
OH
OC2H 4
OH
OC2H 4
OH
OC2H,OH
OH
OC2H,OH
OCHOH
OH
OC2H 4
OH
OH
OC2H 4
OH
OC2HOH
OH
POMS POMS OH POMS POMS OH OH OH POMS POMS OH POMS POMS OH POMS OH OH POMS POMS OH POMS POMS OH POMS ATES ATES OH ATES ATES OH OH ATES OH ATES ATES OH ATES ATES OH OH ATES OH ATES ATES OH ATES ATES OH OH ATES OH ATES ATES OH ATES ATES OH OH OH ATES ATES OH ATES ATES OH ATES OH OH ATES ATES OH ATES ATES OH ATES PTES PTES OH PTES PTES OH OH PTES OH PTES PTES OH PTES PTES OH OH PTES OH PTES PTES OH PTES PTES OH OH PTES OH PTES PTES OH PTES PTES OH OH OH PTES PTES OH PTES PTES OH PTES OH OH PTES PTES OH PTES
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
S :/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specificaon//03.O5.05
S
47 952 K'-1 K-l K 1 2 953 K2-1 K 1 1 K3-1 954 K''I K 3-1 955 K211 K-l K3-1 956 K''1 K' K3-'_ 957 K2-1 K-l K 3 -1 958 K' 1 Kl-l K 3 -1 959 K2-' K''l K 4 -1 960 K''i K' 1
K
4 961 K2-1 K'- 1
K
4 -3 962 K'-l K 4 -3 963 K2-' g 1 1
K
2 -1 964 K2-1 K K 3-1 965 K 2 K'I' K 3- 966 K2-1 K-l K 3 967 K2-' K''l K 3 -1 968 K2-' K'-l K 3 -1 969 K2-' K 3-1 970 K2-' K 3-1 971 K2-' K-l IC 3-1 972 K2-1 K 3-1 973 K(2-1 K3-1 974 K 2-1 1-l K3-1 975 K2-' K 3-1 976 K2-1 K'-l K 3-1 977 K'2- K-l K 3-1 978 g 14 K 979 K'1- K 3 1- 980 K'1- K3-1_ 981 K> 1 K 982 K 1 1 K'-l K 3-1 983 Kl>' K 3-1 984 K' K>-1 K 3- 985 K>'1 K>'1 K3- 1 986 K-l K 3-1 987 K' K' K 3-1 988 K' K' K 3-1 989 K>'I K t' 3- 990 K' K'-l 4- 991 K2-1 K K 4 -1 992 K'2- K'-l K 4 -1 OC2HOH
OH
OH
PTES OH PTES SH SH OH SH SH OH POMS SH OH 0 0 0 0 0 0
OH
OH
OH
SH
SH
SH
SH
SH
SC
2
H
4 OC tBu Po1IS SH OH H 00 SC2H 4 OC (0)
SCH
4
C
2
SC
2
E
4
C'
8
SC
2
E
4
C'
4
SC
2
E
4 C"o S (CE 2 3C 2 1 S (CE 2 3 C18 S (CE 2 3 C14 S (CE 2 3C"O S (CE 2 4 C20 S (CE 2 4 C18 S (CE 2 4
C'
4 S (CE 2 4 C10
SC
2
H
4
C
2 1
SC
2
E
4
C'
SC
2
H
4 C14
SC
2
H
4 C"1 S (CE 2 3C20 S (CH2) 3
C'
8 S (CE 2 C14 S (CE 2 3C'" S (CH2) 4 C20 S (CE 2 4
C'"
S (CE 2 4
C'
4 S (CE 2 4
C'
0
SC
2
H
4
C
2 0
SC
2
H
4 Cl'
SC
2 4 C1 4 Ph ATES SE ATES SE OH OH OH SE OH OH OH SE OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH OH OH OH OH OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S :/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 48 993 K 1 1
SC
2
HC
10 OH OH OH H 0 0 994 K4- S (CH 2 3 C20 OH OH OH H 0 0 995 K2-' S (CH 2 C OH OH OH H 0 0 996 K 2 K' K' 0 S (CHI) 3 C1 4 OH OH OH H 0 0 997 K2- 1 1 K4-1_ S(CH 2 3
C
1 0 OH OH OH H 0 0 998 K2-1 K4-' S(CH 2 4 C20 OH OH OH H 0 0 999 K2-' K4-' S (CH 2 4 CI8 OH OH OH H 0 0 1000 K 2- K 4 S (CH 2 4C14 OH OH OH H 0 0 1001 K2-' K l S (CH 2 4 C10 OH OH OH H 0 0 1002 K 1 1 I K 1 1 K 4- SC 2
H
4
C
2 OH SH OH H 0 0 1003 Kl' K 4-1 SC 2
H
4
C
08 OH SH OH H 0 0 1004 K''K SC 2
H
4
C'
4 OH SH OH H 0 0 1005 K4-1_ SC 2
H
4
C
1 0 OH SH OH H 0 0 1006 K-IC Kl-l K4- S (CH 2 3 C21 OH SH OH H 0 0 1007 K''K S (CH 2 3 C1 8 OH SH OH H 0 0 1008 K 11
K
1 4 S (CH 2 3
C
1 4 OH SH OH H 0 0 1009 K' 1
K
1 K4-' S (CH 2 3 C1 0 OH SH OH H 0 0 1010 K' 1 K''K4 1 S (CH 2 4
C
20 OH SH OH H 0 0 1011 K 1 K- S (CH 2
C
1 8 OH SH OH H 0 0 1012 K'- 1
K
1 -l K4-1- S (CH) C 1 4 OH SH OH H 0 0 1013 1(1 S (CH 2 4
C
0 OH SH OH H 0 0 1014 K 2 1 Kl-l K4-3 SC2HC 2 OH OH OH H 0 0 1015 K 2
K
1 -l K 4 3 SC2H4C' 8 OH OH OH H 0 0 1016 K 2 Kl''K -K4 SCAHC 1 4 OH OH OH H 0 0 1017 K2- 1
K
1 1
K
4 3 SC2HC 1 0 OH OH OH H 0 0 1018 K 2 1 4 3 S (CH2) C 2 0 OH OH OH H 0 0 1019 K 2
K
1 1 K-3- S (CH 2 3 C1 8 OH OH OH H 0 0 1020 K2-1 K11 K -3 S (CH2) 3C14 OH OH OH H 0 0 1021 K2-' K-l K4-3- S (CH 2 3CIO OH OH OH H 0 0 1022 K2-' Kl-l K 4 S (CH 2 4 C20 OH OH OH H 0 0 1022 KICK- 3 S (CH 2 4 C18 OH OH OH H 0 0 1024 K 2 1
K'
1 K- S (CH 2 4C 1 4 OH OH OH H 0 0 1024 K2- 1
K
1 -l K-3 S (CH 2 4
C'
4 OH OH OH H 0 0 1026 KCl KCl K4- 3
SC
2 C21 OH OH OH H 0 0 1026 K 1 K' K4-3 SC2HC 2 OH SH OH H 0 0 1027 K'- 4 K''I K 4 -3 SC 2
H
4
C
1 8 OH SH OH H 0 0 1028 K 11 l Kl-l K4-3 SC2HC 1 0 OH SH OH H 0 0 1030 K 1 K K 3 S (CH2) 3C20 OH SH OH H 0 0 1031 KIC1 Kl-l K4-3 S (CH 2 3 C18 OH SH OH H 0 0 1032 KIC1 Kl-l K4-3 S (CH 2 3 C14 OH SH OH H 0 0 1033 ICICKl 3 S (CH 2 3 C10 OH SH OH H 0 0 S :/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification/03.050 0 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 K1-' K1-1 K 4 -3 K1-1 K1-' K 4 -3 K1-' K1-' K 4 -3 K'21 K1-1 K2'1 K2-1 Kl-l K 2-1 2-1 K 1-1 K -1 K2-1 1-l K2-1 2-1 K K2-1
K
2 -1 K'1-
K
2 -1
K
2 -1
K
2 -1
K
2 -1 K'-l K 2 -1_
K
2
K
1 -l K 2 -1
K
2 K'-l K 2 -1
K
2 K'-l K 2
K
2 K1' Kl-' K 1 'i Kl'
K
2 Kl 1
K
2 1'
K
1 '1 K''i
K
1 '1 K K''l K
K
1 '1 K' K K' K' 1 1-1 11 0 S (CE 2 4 C20 S (CE 2 C1 8 S (CE 2 4
C'
4 S (CE 2 4 C10
SC
2
E
4 C2
SC
2
E
4
C'
8
SC
2 4 C14
SC
2
H
4
CI"
S (CE 2 3
C
2 0 S (CE 2 3C' 8 S (CE 2 3
C'
4 S (CE 2 3 C"0 S (CE 2 4
C
2 0 S (CE 2 4
C
8 S (CH 2 4 C14 S (CE 2 4
C"'
5C 2
H,C
2 0
SC
2
E
4
C"
SC
2
H,C'
4
SC
2 H,C10 S (CE 2 3C 2 0 S (CE 2 3C' 8 S (CE 2 3C" 4 S (CH 2 3 C10 S (CE 2 4
C
20 S (CE 2 4 C1' 8 S (CE 2 4 C1 4 S (CE 2 4 C10 49 OH SE OH SE OH SE OH SE OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH SE OH SE OH SE OH SE CE SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE SH OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
SH H SH SH SH SH SH SH SH SH H SH E SH E SH H OH s H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 0 1 K 1- K 1- K C K1-1 SC 2
E
4 C21 K''1 K''1 SC 2
H
4
C'
8 K K SC 2
E
4
C'
4 K''i K''l K SC 2
E
4 C10 K 1- K1-1 KlC KlC S(CE 2 3 C20 K1-' K'1 S(C 2 3
C'
8 K1-' K K S(CH2)C 1 4 K' K 1 1 l K'l 1
S(CE
2 3
C
1 K1-1 Kl-l K' 1
S(CE
2 4
C
2 0 K1-' K 1 1 K1-1 K'-l S(CH 2 4
C
1 8 K'-1 K K1-1 K' 1
S(CH
2 4
C'
4 K-l K-l K''l S(CH 2 4
CI
K
2 1 EOH S :/Chemical/Sankyo/FP200343/FP00343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115
K
2 -1 K-l K3-1- K2-1 K1*l K 3 -1
K
2 1 K1-l K3-1
K
1 1' Kl-l K 3 1 K' J 1
K
1 1 K 3-
K
1 1 Kl-l K 3 1
K
2 1 1 K-l K 4 -1
K
2 -1 K'K 4-'
K
2 1
K
1 I-1 Kle 1 K-l K 4-1 -1 K 1 1
K
1 i4-1 K 1 1 Kl-l K4-1
K
2 -1 K- 1 i 4 3
K
2
K
1 -l K4-1
K
2 K 1-1 I-3
K'
1 K1-1 K4-3
K
1 1
K-
1 K4-3 K'-1 K 1 K4-3 K 1-1 K -1 K4-3 K2-' K- 1 K2-1
K
2 1
K
2 1
K
2 -l K2-1
K
2 Kl-l K 2 -1 K 121 K'1" 1 K K 1 1 K K 1 1 K'1" 1 Kd-i K'-l
OC
2
H
4
OH
O (CR 2 3
OH
O (CR 2 408
OH
OC
2
H
4
OR
O (CR 2 3 0H O (CR 2 4 0H
OH
OC
2
H
4
OR
O (CR 2 3 0R O(CH2) 40H
OH
OC2H 4
O
O (CR 2 308 O (CR 2 40H
OH
OC
2
H
4
OR
O (CR 2 308 O (CR 2 40H
OH
OC
2
H
4
OR
O (CR 2 308 O (CR 2 408
OH
OC2H4OR O (CR 2 3 0H O (CR 2 408
OH
OC2H 4
OR
O (CH 2 308 O (CR 2 4 0H 50 OH OR OH OR OH OR SH SR OH SR OH SR OH SR SH OR OH OR OH OR OH OR SH SR OH SR OH SR OH SR SH OR OH OR OH OR OH OR SH SR OH SR OH SR OH SR SH OH OH OR OH OR OH OR SH SR OH SR OH SR OH SR SH SR OH SR OH SR OH SR OH OR OH OR OH OR OH OR OH OR OH OH OH OH S R 0 OH OH 5 H 0 OH OH S R 0 OH OR S H 0 OH OH S H 0 OH OR S R 0 OH OH S H 0 OH OR S H 0 OH OH S R 0 OH OH S H 0 OH OH S H 0 OH OH 5 H 0 OH OH 5 H 0 OH OH S H 0 OH OH S R 0 OH OR S R 0 OH OR S H 0 OH OH 5 H 0 OH OR S H 0 OH OH 5 H 0 OH OH 5 H 0 OH OH 5 H 0 OH OH 5 H 0 SH OR S H 0 SH OR S H 0 SH OR S R 0 SH OR S R 0 SH OR S H 0 SH OH S H 0 SH OH S H 0 SH OH 5 H 0 SH SH OH S R 1 SH SR OH S H 1 SH SR OR S H 1 SH SR OR S H 1 OH 5C 2 4
C
2 1 o H 0 OH SC 2
R
4 0 H 0 OH SC 2
H
4 C" 0 H 0 OH SC 2
R
4 C"o 0 H 0 OH S(CH2) 3 C2 0 H 0 OH S(CR 2 3 C" 0 H 0 P89346/FP-200343/gds-mg/specification/03.05.05 K1-1 K' 1 K' K1-1 OH Kl-l Kl-l Kl-l Kl-l OC2HOH K'1 K1- O(CR 2 3 0H K'-1 Kl'" K-l O(CH 2 4 0R
K
2 1 K1-1 K 3 OC2H40H K2-1 K1-1 K-1 OC2H40H K2-1 K1- K 3
OC
2 ,rOH K2-1 K 3 1 OC2H 4 0
H
K2- Kl-l K 3-1 O(CR 2 3 0H
K
2
K
3 1 0 (CR 2 30H S:/ChemicaIISankyo/FP200343/FP200343s.do
S
51 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156
K
2 K'l' K 3 1
K
2 K1-1 K3-1 K 2 K1-' K 3 -1
K
2 K1- K 3-1
K
2 -1 K1-1 K 3 -1
K
2 -1 K1-1 K 3 -1_
K
3 K1-' K'-l K3- K K'-l K 3 1 K'-1 K 3- K1-' K'1 K 3-1 K1-' K 3- K'-1 K 3 -1 K' K'-l K 3 -1
K
2 -1 K1-1 K -1
K
2 -1 K1' K -1
K
2
K
2 -1 K K -1
K
2 -1 K-l K 4 -1
K
2 Kl-l K 4 -1
K
2 Kl-l K 4 -1 K2-1 K-l K 4 -1
K
2 Kl-l K 4 -1 K K''l K 4 1 K2-1 11lK K 21Kl- K 4 1
K
2 1 K 4-1 K1-' K'-l K4-' K'1' l- K' I4-1 K1-1 K''l K 4 1 K''l K 4 1 K ll K ll o (CH 2 30H o (CH2) 30H o (CH2) 4 0H o (CE 2 4 0H o (CE 2 4 0H o (CH 2 4 0H OC2H, 4
OH
OC2H40H OC2H 4
OH
OC2H 4
O
O (CE 2 30H O (CE 2 30H o (CE 2 30H O (CE 2
,O
O (CE 2 4
O
O (CE 2 4 0H O (CE 2 4 0H o (CH 2 4
OH
OCp 2 4
OH
OC2H 4
OE
OC2H, 4
O
OC2H40H O (CE 2 3 0H o (CE 2 3 0H O (CE 2 30H O (CE 2 30H O (CE 2 0O O (CE 2 4 0H O (CE 2 4 0H O (CE 2 4 0H OC2H40H OC2H40H OC2H40H OC2H40H 0 (CH2) 30H 0 (CH2) 30H O (CE 2
,O
O (CE 2 3 0H O (CE 2
,OE
o (CH 2 4 0H O (CE 2 4OH o (CH2) 4 0H
S(CE
2 ),C1 4 0 S(CH2) 3
C
0 0
S(C
2 4 C 200 S (liO 4180 S(CH2) 4 C14 0
S(CE
2 4
C'
4 0
S(C
2 4 C2 0
SC
2
E
4 C" 0
SC
2 HC'4 0
SC
2
E
4 C'1 0 S(CH2)3C 0 0 S(CH,) C" 0O
S(CE
2 )3C14 0
S(CE
2 3
C"
8 0
S(CE
2 )4C20 0 S(CE 2 XC18 0 S(CH2) 4 C4 0
S(CE
2 4 CO 0 SC2E 2 C2 0 0 SC2H, 4 C' 0 SCEC14 0
SC
2
H
4 C'o 0 S(CH2) 3c20 0 S(CH2)3C18 0 S(CH2) 3C14 S(CH2)3C 0 S(CH2) 4 C 0
S(CE
2 )4C is0
S(CE
2 )4C'1 0
S(CE
2 CC' 0 SC2HC20 0 SC2H 4 C" 0 SC, C 1 0 SC2H 4 C' 0 S(CH2)3C2 0 0 S(CH2)3C 0 S(CH2)C'4 0 S(CH2)C' 0 0 S(CH2)4C20 0
S(CE
2 4 C i 0 S (C 2 4
C
4 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 S :/ChemicaUSankyo/FP20343/FP200343s .doc P89346/FP-200343/gds-mg/specificadon//03.05.05 0 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 K 4-1 2-1 K 1-1K 4 3
K
22 K1- 2
K
4 -3
K
23 K1- 3 K4 -3
K
24 K1- 4 I-3
K
2 -1 K1-1 I 4 -3 K2-2 K1-2 K-3 K2-3 K 1-3 K-3
K
24 K1- 4
K
4 -3
K
2 -1 K4-3
K
22 K1- 2 i-3
K
2 3 K1- 3 K-3 K2-4 K1-4 K4-3 K-1 K K4-3 K1-1 K 4 -3 K1-1 K 4 -3 K1-' K4-3 K'-1 K4- 3 K1-' K1-' K 4 3
K
4 -3 K1-' K 4 -3 K'-1 K''1 K4-3 Kl-l K 1-1 K4-3 K1'1 K1-1 K 42
K
2 K1-1 K -3 *2-1 1-l K 2-1_ K2-1 Kl-l K 2-1
K
2 K1-1 K 2 -1 l-1 Kl-l K2-1
K
2 K-l K 2-1
K
2 K'-l K 2 -1
K
2 -1 K'l' K2-'
K
2 -1 Kl-l K 2 -1
K
2 K'-l K 2 -1 21l 1-1 2 11 K K
K
K1-1 K1-' K 1- K K 0(CH 2 4 0H 0C 2
H
4 0H 0C 2
HOH
OC
2
HOH
OC
2
H
4
OH
0 (CH 2 3 0H o (CH2) 30H 0 (CH2) 3 0H 0 (CH2) 30H 0 (CE 2 40H 0(C 2 4 0H 0 (CH2) 40H o (CE 2 40H 0C 2
H
4 0H OC2H40H OC2HOH OC2H,OH 0 (CH2) 30H 0 (CH2) 30H 0 (CH2) 30H 0 (CH2) 30H 0 (CH2) 40H 0(CH2)40H 0 (CE 2 30H 0 (CE 2 40H OC2H40H OC2H,OH OC2H40H OC2H40H 0 (CE 2 30H 0 (CE 2 30H 0 (CH2) 30H 0 (CH2) 30H 0 (CE 2 40H 0 (CE 2 40H 0 (CE 2 40H 0 (CH2) 40H
OC
2
H
4
OH
OC2H40
OC
2
H
4
OH
OC2H 4 0H 52 OH SE OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH SE OH SE OH SE OH SH OH SH OH SE OH SE OH SH OH SH OH SE OH SE OH SE OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH SH OH SE OH SH OH SE
S(CH
2 4
C'
0 0
SC
2
E
4 C20 0
SC
2
H
4 Cl' 0
SC
2
H
4 C" 0
SC
2 4 C10 0 S(CH2) 3c20 0
S(CH
2 3 Cs 0
S(CH
2 3 C14 0
S(CH
2 3
C
4 0
S(CH
2 )4C2 0 0
S(CH
2 4 Cl 0
S(CH
2 4 CC4 0 S(C8 2
)C'
4 0 S (CH 2 4 C 1 0 0
SC
2
H
4 C 200
SC
2 4 C 1 8 0 SC,8 4
C'
4 0
SC
2 H,C" 0
S(CH
2 3 C20 0 S(C8 2 3 C" 0 S(C8 2 3
C
4 0
S(CH
2 3 C 1 0
S(CH
2 4 C20 0
S(CH
2 4 C" 0
S(CE
2 4
C'
4 0 S 4 C' 0
SC
2
H
4 C 20 0
SC
2
H
4 C18 0
SC
2
H
4 C1 4 0
SC
2
H
4
C'
0 0 S(CH2) C20 0 S (CH 2 3 C" 0
S(CH
2 3
C
1 4 0
S(CH
2 3 C" 0 S(C8 2 )C42 0 0
S(CH
2 4 C i 0
S(C
2 4 C'1 0
S(CH
2 4 C1 0 0 SC2H 4 C'20 0
SC
2
H
4 C" 0
SC
2
H
4 C1 4 0
SC
2
H
4 CO 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 S :/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 0 53 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 K K' K 0 (CH 2 3 0H
K
11
K
1 1
K
1 1 0(CH 2 3 0H
K
1
K
1
O(CH
2 3 0H
K
1 1
K
1 1 K' 0(CH 2 3 0H K1-1 K1-1 K 1 l 0 (CH 2 4 0H K 1 1 Kl 0 (CH 2 4 0H
K
1 1
K
1 1
K
1 1
O(CH
2 4 0H K' K 1 K' 0 (CH 2 4 0H K1- 1 K-l K1-1 K 1 -l OC 2
H
4
ON
K
1 1
K
1 -l K-l OC 2 H40H K'1 K-l K 1 Kl-' OC 2
H
4
ON
K
1 K'-l K 1 1 K-l OC 2
H
4 0H K1-' K'-l K'-l K 1 -l 0(CH2) 3 0H K-l K 1 -l K 1 1 l K 1 0 (CH 2 3 0H K1- 1 K1-1 K1- 1 0(CN 2 3 0H K 1
K
1 1 K 1
K
1 0(CH 2 3 0H
K
1 -1 Kl 1
K
1
K
1 -l 0(CH 2 4 0H
K
1 1 K1- 1
K'
1 1 K' 1 0(CH 2 4 0H
K'
1
K'
1 K'l 0 (CH 2 4 0H KIIK1-1 K 1-1 K 1-1 0(CH2) 40H 2-1 Kl-l 3-1 2-1 Kl-l K 3-1
K
2 1
K
3 1
K
2
K
1 -l K 3 -1 K2-1 K l K 3-1 K-1 Kl-' K3-1 l21 K11 K3-1 K2-1 Kl-l K 3-1
K
2
K
1 -l K3-1
K
2 1
K
1 -l K3-1 K2-1 K 1 -l K3-1 K2-1 Kl-l K3-1 K1- 1 K'-l K 3 1 K 1- K 1 -l K 3 1 K1-1 K 1 -l K 3 1
K
1 1 K1- 1 K3-1
K
11 K-l K 3 -1_
K
1 -l K 3
K-
1 1
K
1 3 -1 K 'l Kl 1
K
3 1
SC
2
H
4
C
2 1
SC
2
H
4 C18
SC
2
HC
1
SC
2
H
4
C
1 0 S (CH 2 3
C
2 0 S (CH 2 3 C"s S (CH 2 3
C'
4 S (CH 2 3
C
10 S (CH 2 4
C
20 S (CH 2 4
C'
8 S (CH 2 4
C
14 S (CH 2 4
C
1 0
SC
2
HC
20
SC
2
HC
18
SC
2
H
4
C
1 4
SC
2
H
4 C"o S (CH 2 3
C
2 0 S (CH 2 3 C18 S (CH 2 3 C14 S (CH 2 3
C
10 S (CH 2 4
C
20
S(CH
2 3 C20 0 S (CH 2 3
C'
8 0 (CH2) 3C 14 0
S(CH
2 3 C1' 0 S(CH2) 4c20 0
S(CH
2 )4Cl' 0
S(CH
2 4 Cl' 0
S(C
2 4 C 100 SH SC 2
HC
20 0 SH SC 2
HC
18 0 SH SC 2 A C 1 4 0 SH SC 2
H
4 C'o 0 SH S(CH 2 3
C
2 1 0 SH S(CH 2 3 C18 0 SH S(CH 2 3
C
14 0 SH S(CH 2 3
C'
0 0 SH S(CH 2 4 C0 0 SH S(CH 2 4 C'1 0 SH S (CH 2 4
C'
4 0 SH S(CH 2 4 C1 0
SC
2
H
4 C20 0
SC
2
N
4 C 1 0
SC
2
H
4 0
SC
2
H
4 C' 0
S(CH
2 3 C20 0
S(CH
2 3 C1' 0
S(CH
2 3
C
4 0
S(CH,)
3 0
S(CH
2 4
C
2 0 0
S(CH
2 4
C
1 8 0
S(CH
2 4
C'
4 0
S(CH
2 4 0
SC
2
H
4 C20 0
SC
2
H
4 C18 0
SC
2
H
4 C4 0
SC
2
H
4 C'o 0
S(CH
2 3
C
2 0 0
S(CH
2 3
C
1 8 0
S(CH
2 3 C4 0
S(CH
2 3 C" 0 S(CH2)4C2) 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 11 11 11 11 11 11 11 11 11 11 11 11 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 S :/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specifcaion//03.05.05 0 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 K1-1 K 11 K3-1 Ki-l K3-1 1-1 K1-' K3-1 2-1 Kl-l K4-1 12-1 K(1-1 14-1 2-1 K1-l K4-1 2-1 Kl-l K 4-1 2-1 K1- 1 2-1 Kl-l K 4-1- 2-1 Kl-l K 4-1 2-1 Kl-l K 4-1 X2-1 K1-1 X4-1 12-1 K1-3 4-1 K2-1 K1-1 4-1 X2-1 K -1 X4-1 1-1 1(1-3 K 4-1 24 1 K 1-1 K-l K 4-1 Ki-I Kl-l K 4-1 K 1-1 Kl- K -1 K 1-1 Kl-l K 4-1 Kl-l Kl-l K 4-1 K 1-1 Kll K 4-1 K 1-1 Kl-l K 4-1 2-1 K 1-1 K4- K2-2 K 1-2 K4-3 K2-3 K 1-3 K4-3 K2-4 K 1-4 K4-3 2-1 K 1-1 K4-3 j-2 K -2 K4-3 K2-3 K -3 K4-3 K2-4 K 1-4 K4-3 K 1-1 K 1-1 K4-3 K -1K- S (CH 2 4
C
18 S (CH 2 4
C
14 S (CE 2 4
C
10
SC
2
H
4
C
2
SC
2
H
4
C
18
SC
2
H
4
C
1 4
SC
2
H
4
C
0 S 3
C
20 S (CH 2 3
C
18 S (CE 2 3 C 14 S (CE 2 3
C'O
S (CE 2 4
C
2 0 S (CE 2 4
C
18 S (CE 2 4
C'
4 S (CH 2 4
C
0
SC
2
H
4
C
2
SC
2
H
4
C
18
SC
2
E
4 C1 4
SC
2
H
4
C
10 S (CE 2 3
C
20 S (CE 2 3
C
1 S (CE 2 3 C14 S (CE 2 3
C
10 S (CE 2 4
C
20 S (CE 2 4 C'i S (CE 2 4
C'
4 S (CE 2 4
C
10
SC
2
HC
2 0
SC
2
H
4
C'
8
SC
2 HC1 4
SC
2
H
4
C'
S (CH2) 3
C
20 S (CE 2 3
C
1 8 S (CE 2 3
C'
4 S (CE 2 3 C10 S (CE 2 4
C
20 S (CE 2 4
C
1 S (CE 2 4 C14 S (CE 2 4
C'
0
SC
2
E
4
C
2
SC
2 H4
C'
54 OH SE OH SE OH SE OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH CE OH OH OH OH OH OH OH OH OH SE OH SE OH SH OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH SE OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH CE OH OH OH OH OH OH OH OH OH SE OH SE S (CH2) 4O18
S(CE
2 4
C
14
C
S(CH
2 4
C
1 0
C
SCA CC20 0
SC
2
E
4 Cl' C
SC
2 E C 4
C
SC
2
E
4
C
S(CH2)C 0
C
S(CE
2 3 C C
S(CE
2 )4C C0
S(CE
2 3
C
14
C
SC,,20
S(CE
2 4 C" C
S(CE
2 4
C
1 8
C
S(CE)C'
4
C
S(CE
2 4
C'
0
C
SC
2
E
4 C20 C
SC
2
H
4 C" C SCA C14 0
SC
2
E
4 Clo C S(CH2) C21 0 S(CH2) 3C" 0 S C 2
C
10
C
S(CE
2 3 C C
S(CE
2 3 C C
S(CE
2 )C C
S(CE
2
)C
1 4 C
S(CE
2 4 C" C SC2H4C 20 0 SC2HC8
C
SCA 14 1 SC2H4C) 4
C
S(CE
2 )C2 0
C
S(CH2) 3CI8 0 S(CH2) C14 0 S(CH2) 3C'O 0 S(CH2)CX S(CH2) 4
C
C
H,
4
C
1 4
C
S(CE
2 )4C 0
C
SC
2 C21 C SC2H 4 CC C 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 S :/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specificatio//03.05.05 0 55 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320
K'
1 1 K1-1 K4-3 K1-1 K1-1 K 4 -3 K1-1 K 11 K4-3 K1-1 K1-1 K4-3 K1*1 K1-1 K4-3 K11 K1' K 4-3 1-1 K 1- K4-3 K1-1 K 1-1 1-3 K1- 1< K -3 K21 K1-1 K2-3- K2-1 K1-1 K2-1 K 2- Kl-l K2-1 K2-1 K1-l K2-1 K 2-1 Kl-l K 2-1 K21 K1 K2-1 2- 11 K2-1 K2-1 Kl-l K2-1 2-1 K1-l K2-1 2-1 K1-1 K2-1 K<1-1 K1-l K2-1 K<1-1 K<1-1 K 1-1 1<1-1 K<1-1 111 K 1-1 K<1-1 11 K -1K1- K- K K11
SC
2 A C 4
SC
2
H
4
C"
S (CH 2 3
C
20 S (CH 2 3
C
1 8 S (CH 2 'C1 4 S (CH 2 3C'" S (CH 2 4
C
20 S (CH 2 4
C
8 S (CE 2 4
C
1 4 S (CE 2 4
C
10
SC
2
HC
2
SC
2
H
4
C
18
SC
2
H
4
C
14
SC
2
HC'
0 S (CE 2 3C 20 S (CE 2 3
C
18 S (CE 2 3
C
14 S (CH 2 3
C
10 S (CH 2 4
C
20 S (CE 2 4 Cl S (CE 2 4
C
14 S (CH 2 4
C
10
SC
2
H'C
2
SC
2
H
4
C
1 8
SC
2
E
4
C
14
SC
2
H
4 C'o S (CE 2 3
C
2 1 S (CE 2 3 C's S (CE 2 3C 14 S (CE 2 3
C
10 S (CE 2
C
20 S (CE 2 4
C'
8 S (CE 2 4
C
14 S (CE 2
IC'
0
SC
2
E
4 C" 0
SC
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S(CH
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C
20 0
S(C
2 3
C
18 0
S(CE
2
C
14 0
S(CE
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C'
0 0 S (CH 2 4 C20 0
S(CE
2 4 C" 0
S(CH
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S(CE
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C
1 0 0
SC
2
H
4
C
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2
H
4
C
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E
4
C
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C
10 0
S(CE
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S(CE
2 3
C
18 0
S(CH
2 3
C'
4 0
S(CH
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C
2 0 0
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2
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4
C
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C
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2
E
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2
C'
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S(CE
2 3
C'
0 0
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S(C
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S(CH
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C
14 0
S(CH
2 4
C
1 0 SH SC 2
H
4 C20 0 SH SC 2
E
4 Cl 0 SH SC 2 A C 14 0 SH SC 2
E
4
C
10 0 SH S(CH 2 3
C
20 0 SH S(CE 2 3
C'
8 0 SH S(CH 2 3 C4 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 .0 1 0 1 0 1 11 11 11 11 11 11 11 K1-1 K1-1 K1-1 K 1- SC2H 0 C21 1<-l Ki KI-' Kl-l SC 2
E
4 Cl' K1 1 K-l k-l SC 2 4 C14 Ki-I Ki-' Ki-l K1<' SC 2
E
4 Clo K1ll K1ll K1-1 K-1 S(CE 2 3
C
20 Kl-l Ki-l Ki-' S (CH2) 3C18 K1-1 K1-1 K-1 K1-1 S(C1 2 3 C14 S :/ChemicaUSankyo/FP200343/FP00343s .doc P89346/FP-200343/gds-mg/specification/03.05.05 56 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 K''1 K 3 '1 K''I K'1 S(CH2) 3 C 1
K'
4 i K''1 K''I K' S (CH 2 4 C 2 0 K''I K''i K'I K'i S (CH 2 4
C'
9 K''I K''1 S (CH 2
IC'
K
1 'i K''i K' S (CH 2 4 C1 0 S (CH 2 3 C1 0 S (CH 2
C
2 1 0 S 4 C' 0 S (CH 2 'C1 4 a S ,C"o 0
K
2 1 K 3 2
K
2 -1 K'-1 K 3 2 K''i K 3 2 K''1 K 3 2 K 2 K''1 K4- 2 K 21KI- K 4 2 K''i K 4 2 K''I K''l K 4 2
K
2 1 K''1 K4 1 -4
K
2 1 K4-4 K''i K 4 4 Ki- Kl K4- K2-1 1- K2-2 K 21K- K2- K''I K 1 2 K''i K 1 2
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2
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K
2 1 K'11 K 3 2
K
2 1 K 1-1 K 3 2 K K 3 2 K1-1 1-1 k3-2 K 21K 11K4-
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4
OH
S:/Chemical/Sankyot'FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification/03.05.05 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 K1-' K'l' K3-'
K
2 -1 Kl-l K 3-1
K
2 -1 K 4-1 1-1 K-l K4-1 K-1 K 11 K4-3 K1-1 K 1- K4-3 K2-1 K1-l K 2-1 K2-1 K1-1 K31-1 K2-1 Kl-l K3-1 K1-1 K 3 -1 K2-1 Kl-l K 4 -1 K-1 K 4 3 K K 1- 1 K'-3
K
2 K1-1 K 2-1
K
2 -1 Kl-l K 3 -1 K K 3 -1
K
2 -1 Kl-l K 4 -1 K''I K 1 K4'-1
K
2 K 1-1K4-3 K1-1 K'-1 K4-3
K
2 K K 2
K
2 1 K1-1 K 3 -1
K
2 K 1- K 3 -1 K1-1 K 3 -1
K
2 -1 K 4 K 1 l' K I4-1 K2- K1-1 K4-3
K
11
K
1 1
K
4 -3 K-1 Kl-l K2-1
K
2 -1 K'1 K 3 -1 Kle' K 3 -1
K
2 -1 Kl-l K 3 -1 K'-1 K 3 -1 K 3- K' K3-1 K-l K-l K 3 -1
K
2 1 K 4 1
K
2 1 K 4 0C 2
H
4 0H
OC
2
H
4 0H 0C 2
H,OH
OC
2
H
4
OH
OC
2
H
4 0H OC2H40 OC2H40
SC
2
HOH
SC
2 4 0H
SC
2 H40H
SC
2
HOH
SC
2
H
4 0H
SC
2 H40H
SC
2
H
4 0H
SC
2
H
4
OH
S (CH 2 308 S (CH 2 30H S (CH 2 30H S (CH 2 30H S (CH 2 3 0H S (CH 2 3 0H S (CR 2 3 0H S (CH 2 308 S (CH 2 408 S (CH 2 408 S (CH 2 408 S (CH 2 408 S (CH 2 408 S (CH 2 408 S (CR 2 408 S (CH 2 408
SC
2
H
4 0H 0C 2
H
4 0H 0 (CH 2 308 0 (CH 2 408
SC
2
H
4 0H OC2H40H 0 (CR 2 308 0 (CH 2 408
SC
2
H
4
OH
OC2H40 57 OH S8 OH S8 OH OH OH S8 OH OH OH SN OH OH OH OH OH S8 OH SR OH OH OH SH OH OH OH S8 OH OH OH OH OH SN OH SR OH OH OH SH OH OH OH SH OH OH OH OH OH SN OH SH OH OH OH SH OH OH OH SH OH OH OH OH OH OH OH OH OH OH OH SN OH SH OH SH OH SH OH OH OH OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
2
H
4 0H 0 H
SC
2 HOH 0 H
S(CH
2 3 0H 0 H
S(CH
2 4 0H 0 H
SC
2
H
4 0H 0 H
SC
2
H
4 0H 0 H
S(CH
2 3 0H 0 H
S(CH
2 4 0H 0 H
SC
2
H
4 OH 0 H
SC
2 HOH 0 H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 58 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 K2-1 K 1-1 K 4-1 K2-1 K1-1 K 4-1 1<2-1 K1_1 K 4-1 1<1-1 K11K4- K<11 1-1 K4-1 12-1 K1-l K4-3 K 2-1 111 K4-3 K2-1 K1-l K4-3 K2-1 K-1 K4-3 1<-1 1<-1 K4-3 KI-I K4-3_ Ki-I Kl-l K4-3_ Ki-I KI-1 K4-3_ 2-1 K11 K 2-1 2-1 K1_1 K 2-1_ 2-1 K1_1 K 2-1 2-1 KI-1 K 2-1_ Ki-i K-1 KI-1 Ki-i K1_1 K1_1 KIIKiiK-1 KiIK-IKo (CH 2 3 0H o (CH 2 4 0H
SC
2
H
4
OH
OC
2
H
4
OH
o (CH 2 30H 0 (CH 2 401
SC
2
HOH
OC
2
H
4
OH
0 (CH 2 3 0H
O(CH
2
OH
SC
2
H
4
OH
OC
2
H
4
OH
O (CH 2 30 0 (CH 2 4 0H
SC
2
H
4 0H 0C 2
H
4 0H 0 (CH 2 3 0H 0 (CH 2 4 0H
SC
2
H
4 0H
OC
2
HOH
0 (CH 2 3 0H 0 (CH 2 4 0H
S(CH
2 3 0H 0
S(CH
2 4 0H 0
SC
2
H
4 OH 0
SC
2
H
4 0H 0
S(CH
2 3 0H 0
S(CH
2 4 0H 0
SC
2
H
4 OH 0
SC
2
H
4 OH 0 S (CH 2 30H 0
S(CH
2 4 0H 0
SC
2
H
4 OH 0
SC
2
H
4 OH 0
S(CH
2 3 0H 0
S(CH
2 4 0H 0
SC
2
H
4 OH 0
SC
2
H
4 OH 0 S (CH 2 30H 0
S(CH
2 4 0H 0
SC
2
H
4 H 0
SC
2
H
4 OH 0
S(CH
2 3 0H 0
S(CH
2 4 0H 0 SH SC 2
H
4 OH 0 SH SC 2
H
4 OH 0 SH S(CH 2 3 0H 0 SH S(CH 2 4 0H 0 OH Li OH L1 OH LI SH OH L1 OH L1 OH L2 OH L2 OH L3 SH OH L3 OH L2 OH Li OH Li OH LI SH OH Li OH LI H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 01 H 11 H 11 H 11 H 11
ON'_
6 0 2 ON'1 6 0 2
ON'
6 0 2
ON'
6 1 2
ON'-
6 0 2 ONI-6 0 1
ON'-
6 0 1 ON'1 6 01 ON1-6 1 1 ON' 6 0 1
ON'
7 0 2 ON1- 7 0 2
ON
1 7 0 2 ON 1-7 2 ON 1 7 0 2 K<1-1 K-1 K1-1 K 11 SC 2
H
4
OH
K<1-1 K<-1 K1-1 K'-1 OC 2
H
4
OH
K 1-1 K-1 K 1-1 K1_1 0(CH2) 30H K 1-1 K -1 K 1-1 K 1-1 0(CH2) 40H K 2-1 K<1-1 3-1 OC 2
H
4 0H K 1-1 K 1-1 11 OC 2
H
4
OH
K 1-1 11 K1-1 OC 2
H
4
OH
K 1-1 K 1-1 K 11 K1-1 OC 2
H
4
OH
K1-1 1<1- K1<1 OH l K3-' OC 2
H
4 0H 11 11 1-1 OC 2
H
4 0H K1-1 11K 11 OC 2
H
4
OH
K 1-1 K -1 K -1 K 1-1 OC2H40H K<1-1 K1-1 K1_1 OH K2-' K11 K3-1- OC 2
H
4 0H K<1-1 K1-1 K<1-1 OC 2
HOH
11 K<1-1 11 OC 2
H
4
OH
K 1-1 K 1-1 OC2H40H K 11 11 K1- OH S :/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification/03.05.05 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 K2-' K3- OCHrOH K I iK11 OC 2
H
4
OH
KI K' K'1- 1 OC2H,OH K''i
K
1
OC
2
H,OH
IC K-IC IC OC 2
H
4
H
K'1 1 K''1 OH
K
2
K
3
OC
2
H
4
OH
K 1 1 K1 K1 1
OC
2
N
4
OH
K 1 1 K1 OCH 4
ON
K''i K' 1 Kl-' OC 2
H
4
OH
K
1 1 K' 1 K1 OH
K
2 K1 OC 2
H
4
OH
K1 1
K
1 K1-' OC 2
H
4
OH
KI K'i K 1 '1 OC 2
H
4
OH
K'-1 K1-' K1-' OC 2
H
4
OH
K
1 K1 K 1 1 OH K2_ 1
K
1 1 K1 OC 2
H
4
OH
K1- 1
K
1 'l K1- OC 2
H
4
OH
K1- 1 K'1 K1 OC 2
H
4
OH
K'
1 K' OC 2
H
4
OH
K1 1 K11 K11 OH K2_ 1
K
3 _1 OC 2
H
4
OH
K>-
1 K>'1 K1-1 OC 2
H
4
OH
K'1 K 11 K 1 OC 2
H
4
OH
g 1 1 Kl>' OC 2
N
4
OH
K 1 '1 K' K' OH K2- 1 K1'1 OC 2
H
4
OH
K1 K 1'1 K 1 OC 2
H
4
OH
K 11 K1-1K 11 OC2HOH K>'i K 1 K-l OC 2
H
4
OH
K
1 K 11 K1 1
OH
K2-1 K1-1 K 3-1 OC 2
H
4
OH
K1 1
OC
2
H
4
OH
K11 K 1 1
OC
2
H
4
OH
K1-1 K 1 1 K1-1 K1-1 OC 2
H
4
OH
K1- 1 K1-1 K1-1 OH K2-1 K11 K3- OC 2
H
4
OH
K 1' K> OC 2
N
4
OH
K> K>1 1
OC
2
H
4
OH
K>1 1 K 1' K' OC 2
H
4
OH
K1_ 1 K 1l K 1 1
OH
K>
1
K>_
1
OC
2
H
4
OH
59 OH OH OH SH SH SH OH SH SH SH OH OH OH SN SH SN OH SH SH SH OH OH OH SH SH SH OH SN SH SH OH OH OH SH SH SN OH SN SH SN OH OH OH SN SH SH OH SH SH SH OH OH OH SN SH SN OH SN SH SN OH ON OH SN SH SN OH SH SH SN OH ON OH SN SH SN OH SN SH SN OH OH
OH
OH
OH
SH ON
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH ON
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH ON
OH
OH
L 0N"' 0 1 L2 ON1-7 o 2 ON 1-7 o i L2 ON 1-7 1 L2 ON1_7 01 L1 ON2-6 0 1 LI ON2-6 0 1 L' ON2- 6 0 2 L 1 ON2-6 1 L' ON2- 6 0 2 L' ON2- 6 0 2 L 2 ON 2-6 o 1 2 ON2-6 0 1 L' ON2-6 1 2 ON2-6 01 L ON2-7 0 2 L1 ON2-7 0 1 2 ON2-7 0 L 1 ON2-7 1 Ll ON2-7 0 2 L 0N2-7 0 1 L2 ON2-7 0 1 L2 ON2-7 L2 ON 2-71 1 L2 ON2-7 0 1 L' ON3-6 0 2 L1 ON3-6 0 2 L1 ON3-6 0 2 L O 3-61 2 Ll ON3-6 0 2 L2 73-6 o 1 L2 ON3-6 0 1 L2 ON3-6 0 1 L2 73-6 11 L 0N3-6 0 1 L ON>- 7 1 1 L ON3- 7 0 1 L' ON> 6 7 0 2 L ON3-7 0 2 1l ON3-7 Ll ON3-7 0 2 L 2 ON3 0 1 S :/ChemicaUSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 50'90'EO//UOEIEO9!30ds/SW-SPS/EtEOOZ-JN9tE68d oopsEt7EooZjq/Et7Eoo7ja/okTmS/I-!u-ioqD/:S T 0 -NO Z'r 1 T 0 -NO Z' 1 z 0 -,No Zr' Z L.-m t"i z 0 L-,NO III Z 0 L-,NO Zr z 0 L-SN 0
III
T 0 -,NO Eli T I -,ZNO El T 0 ,I T 0 -,NO ,I* T 0 -,NMO Zr' z 0 9 sNO r Z T -~SNO r'i z 0 -,NO ~I z 0 -,NO 'r' Z 0 ,NO ,'I T 0 -,NO 'r 1 T T Z' 1 1t 0 L-X 0 Er' T 0 L-INO E'I *T 0 L-N 0 Zr' Z 0 -NO Zr' Z T L-tNO 1r Z 0 L 4
I
0 Zri Z 0 L- 0 'r.i Z 0 LIN r" T 0 Z"i T T -,NO El T 0 -,NO r'i 1 0 ,NO Z"i T 0 ,NO Zri Z 0 'rI Z T -,NO III~ Z 0 -,NO r'i Z 0 TrI Z 0 1 ri T 0 L-INO 'I T T L-ENO Zi T 0 L-N 0 Zr T 0 L.CNO Z"i Ho HS HO HO Ho MS HO HS HS HO HS HO HS HO HO HO HS HO MS MS HO HS Ho MS Ho HO HO HS HO HS HS Ho HS HO HS HO Ho HO HS HO0 MS HS Ho MS HO HS HO HO HO HS HO MS HS HO HS Ho MS Ho HO Ho HS HO HS HS HO HS HO HS HO HO HO HS HO MS HS Ho HS HO HS 14O HO Ho HS HO MS MS MO MS HO HS MS MO MO HO MS MS MS MO MS MS HS MO HO MO MS MS MS MO MS MS MS MO MO MO MS MS MS MO MS MS MS HO MO HO MS MS HS MO MS MS MS MO MO MO HS MS MS MO MS MS MS MO MO HO MS MS MS MO MS MS MS MO MO MO MS M4S HS MO MS MS MS MO MO MO MS MS MS MO MS MS MS HO 09 HOVHZZ T- N Z Z>T HO0 I-EM T-TNI -zm HO~ZO T-T I-T T- MOV00~ I-Zm '-Pm T-TM HO'HMZ)O ITCM I-TH Z-ZM MO'Z3 T-Zm I-TM I-z HOMZO I-Tx T-TN I-T MO'HZZDO II T-IM I> -ZM H OZ3 T-T IT>M Z-TN HO'HZZDO I-EN ITM~ I- Ho~MD Z._ZX T-Tx I-TM MON~Z0O tIt>m Z113 I-TM HO3 I-I I- T-Tm MO'MZZDO I-EN I-TN T-t> 1 HOMZO T-ZTN T-TN ZTZ>M HO'HM:)O I- ZIE>M I-TH I-T MOHZ) T-Tx ZIZ>N ZIZ> HO'HZZ)O Z-Tm T-NII HO'HZZ)O I-E ZTZ>N IzmT MO I-IN~ ZTZ> T-ZN HO'Hz:)O I- ZIZ>! T-NI HONMZOO I-PM ZIZ>I ZI-T HO'HZ:DO I Z-0 1 -NTzm MO ZM -t I Z T> -ZTM MO'HzZo ~m MOI'Mz:O ZIt>M ZT>M T-TM H'O0 IE>N I-ZN T-ZN HO I-T I-I T- MOI.MZZO ZTZ>M I-ZTm ZTZ> OIZO I-I T-m IT
SZST
ZgT
LZST
ZZST
TZST
OZST
61ST 8151
LIT
91ST 5151
I'TST
LIST
ZTST
1151 0151 6051 8051 L0ST
ST
OST
LOST
ZOST
TOST
OOST
66 T L6 T 96 T 96t7T 6 T L6 'T Z0T T66'1 06' T 68 'T 828'I L8 T 98 T S8 'T 90O&OEO//UO~!93ds/W-SP/E,EOOZ-dA9VE68d DOP'SEt7EOOZCL4/Et7coozdNofquLSA-!Lugqo/:s 1 0 -LNO zi HO HS HS HS HO'HzDO I-I I-MIT 99£ T 1 0 -,NO ri HO HS HS HO HO'HzDO I-NTI -m 9T 1 0 -,NO z'I HO HO HO HO HO'HzDO I-XIINIz V99T Z 0 -,NO I HO HS HS HS HO T-mTINII E9ST Z I L-LNO 'I HO HS HS HS HO HOVHOO ,X ,X Z9 Z 0 LLN Tri HO HS HS HS HO'H'ZDO I-NTImIT T99T Z 0 L-LmO r'i HO HS HS HO HO'HzOO I-NII -3 0991 Z 0 L-LNO Tri HO HO HO HO HObHOO ESSI-I -Z 69 1 0 9-LN 0 z'i HO HS HS HS HO T-.
1 -M I-TN 8SST I I 9-LN 0 Z' HO HS HS HS HO HO'HzOO I-IM -IM I- -I LSST 1 0 9-LN'O Z'i HO HS HS HS HOVO~O T-I II t-I 99ST 1 0 9 -LNO ri HO HS HS HO HOV'HOO I-T T-TH I-I SSS 1 0 9 -LNO z'i HO HO HO HO HO'HZDO I T-I I-?I i'££T Z 0 9 .LNO ri HO HS HS HS HO T-T I-T £££ICS Z I 9 -LNO r'i HO HS HS HS HO HONHZOO ITA! I-IN IT ST Z 0 9-LNO !r HO HS HS HS HO'HZ0O I-I I-I IM ISST Z 0 9 -LNO gri HO HS HS HO HO"H'ZDO T-T T-T I-T Oqg Z 0 9-LN 0 'I HO HO HO HO HO'HZZO I-E T-T T- 6'v£I 1 0 LINO' 1 z'1 HO HS HS HS HO TXII T-T B ST I I LINO ri HO HS HS HS HO HO'HZZ0o T-TN I-I I-I T-I 091 1 0 LI 9 NO rl HO HS HS HS HOVOZO I-T t-Im ,x 9 ST 1 0 LIgNO zri HO HS HS HO HOVHOO T-I I-NIIM S 1 0 LIgNO zri HO HO HO HO HO'1H'O I-NI-N MZN S Z 0 LINO r'1 HO HS HS HS HO T-I T-T T-N C Z I LI 9 NO 'ri HO HS HS HS HO HOVHOO tIt>N I-I T-TN I-I Z Z 0 LINO" r'i HO HS HS HS HOVOZO T-mIm T ST Z 0 LINO 0 ri HO HS HS HO HO'Hz:O I-MTINII O ST Z 0 LINO rI HO HO HO HO HONHZDO I-E T-I I- 6ES1 1 0 9 1NO /1i HO HS HS HS HO T-mITMTT 8CST I I I~NO ri HO HS HS HS; HO0 HO'HZOO t-tM I-T T-M LEST I 0 I.NO r'i HO HS HS HS IHz:)O -Pt -IN MI 9CST 1 0 9 9 N0 'i HO0 HS HS HO HO'HZZO I-T T-T>N T-tN SCSI 1 0 9 1NO ,I HO HO HO HO HO'HZO T-m _IM T~z CgT Z 0 I.~NO III HO HS HS HS HO0 I-H MT I-T EST Z 1 9 1NO Tri HO HS HS HS HO HOVHDO I-I T-I IT II ZEST Z 0 9 1~NO TrI HO0 HS HS HS HO'Hz3O I-T I-T I- TEST Z 0 9 1.NO ~I HO HS HS HO HOVHOO IT T-I I- OESI Z 0 9 1NO 'I HO HO HO HO HOVHOO I-E T-T T- 6ZST 1 0 L-NO zi HO HS HS HS HO I-IN I-T T-TN 8ZST IIL-S~NO ri HO HS HS HS HO HO'HZDO 1 -12 I-I I-I LZST I 0 ,~NO zrI HO HS HS HS HONHOO 1-1 T-IN I-m 9ZST T9 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607
K
1 1
K'
4
K
1
K'
1
OC
2
HOH
K K' K OH
K
2 1
K'
1
K
2
OC
2
HOH
Ki K 1 1 K'i OC 2
H
4
OH
K 1 -1 K'- 1
K
1
OC
2
H
4
OH
K
1 '1 K 1 _1 OC 2
H
4
OH
K
1 1 K1 K 1 1 OH
K
2 1
K'
1
K
3
OC
2
HOH
K1 K 1 1 K1 1
OC
2
HOH
K 1-1 K1_ 1 K''l OC 2
HOH
K 1 1 K 1- K 1- K1_1 OC 2
HOH
K
1 1
K
1 1 K1 OH
K
2 K'-l K 3
OC
2
H
4
OH
K
1 1
K-
1
K
1
OC
2
HOH
K
1 1 K1- 1
K
11
OC
2
HOH
K K 1 1
OC
2
H
4
OH
K
1 K 1 1 K' OH
K
2
K
1 1 K 3 1
OC
2
HOH
KI K'I K'i OC 2
H
4
OH
K
1 'i K'I K'I OC 2
HOH
K
1 1 K K 1 1
K
1 1
OC
2
H
4
OH
K
1 1 K1 K1 1
OH
K
2 1
K
1
K
3 1
OC
2
HOH
K11K11K 11- OC2HOH K -1 OC2H,OH
K
1 1 i K' 1 K-1 OC 2
HOH
K 11 11 K1- OH K 2-1 K1-1 K 3-1 OC2HOH 11 K 11 OCOH K KI Ki OC 2
HOH
K
11
K'
1 1 K 11
OC
2
H,OH
K
1
K-
1
K
11
OH
K2-' OC 2
HOH
K K 1 K OC 2
H,OH
K K K OC 2
H
4
OH
K
1 1
K
1 1 i Kl 1
OC
2
HOH
K-
1
K'
1 K'1 OH
K
2 1
K-
1 I K 3 -1 OC 2
H
4
OH
K''i K''i OC 2
H,OH
1 1 K1 1 K1 OC 2
HOH
K-1 K'_ 1
OC
2
H
4
OH
62 OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH SH OH OH OH SH SH SH OH SH SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH
OH
OH
OH
OH
SH OH 2 7-7 L ON 1 1 L2 ON7-7 01 L1 0 1 L1 ON8-6 02 LI 0 2 1 86ON'-' L 1 0N'_ 0 2 L' ON 8 6 0 2 L2 ON' 6 0 1
L
1
ON
8 6 0 2 L1 ON 8- 0 1 L2 O86 L 0N 0 1 L 1 ONI-7 0 2 L2 ON8-7 08 L2 ON'_7 0 1
L
2
ON
8 -7 1 1
L
2
ON
8 -7 0 1 L 0N9_6 0 2 L ON 8 7 0 2 Ll ON9 7 0 2
L
1
ON
8 7 1 2
L
1 ON9 7 0 2 L2 8N967 L 2 ON 96 o 1 L2 ON96 0 1 L2 ON6 1 L2 ON9-6 0 1
L
2 1 ON9 7 0 1 L I ON 9- 0 2 2 7ON9- 0 2 LI ON9 1 1 L1 ON 9-70 1 L' ON9-7 0 2 L ON' 0 2 L1 O9-6 0
L
1
ON
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OC2HOH OC2H4OH OC2HOH OC2H4OH OC2HOH OC2H4IOH OC2H4OH OC2H4OH 66 OH SH OH SH OH SH SH SH SH SH SH SH SH SH SH SH OH SH OH SH OH SH OH SH OH SH SH SH SH SH SH SH SH SH SH SH OH SH OH SH OH SH OH SH OH SH SH SH SH SH SH SH SH SH SH SH OH SH OH SH OH SH OH SH OH SH SH SH SH SH SH SH SH SN SH SH OH SN OH SH OH SH
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3 6 01 1781 1(21 1(l-l OC2HO ON SN SN SN L 2
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3 6 01 1782 Kl11 OCHON ON SN SN SN L 2
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3 6 0 1 1783 1<2-1 1(1-11(2-1 OC2HON ON SN SN SN L 2 0ON 3 6 01 1784 1(2-1 K1-l K(3-1 OC2HON SN SN SN SN L 2 ON 3 6 0 1 1785 K(3-1 OC 2
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4 ON SN SN SN SN L 2
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3 6 0 1 1786 1(2-1 Kl11 1(41 OC 2
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4 ON SN SN SN SN L 2 ON 3 -6 0 1 1787 OCN 4 ON SN SN SN SN L 2
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3 6 01 18 K2-1 11- K21 C2,H SN SN SN SN 2 O 3-6 1 1789 K1( 13-1 OC2HON ON SN SN SN L 1
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6 0 2 1790 K(11 K3- ON, ON SN SN SN L 1
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4 6 0 2 1791 K(2-1 KllK OCN 4 ON ON SN SN SN L 1 0N 4 6 0 2 1792 K1( Kl( OCHOH ON SN SN SN Li ON 460 2 1793 Kl11 1(2-1 OCN 4 0N ON SN SN SN L O 4-6 0 2 1794 (2-1 K3-1 OC 2
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4 0N ON SN SH SN Ll ON 5-6 0 2 S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specificabon//03.05.05 68 1813 OCE 4 OH OH SH SE SH L 0 2 1814 K 2-1 K 1 1 K 3 1
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5 6 0 1 1822 K K1K4- OC 2 HOE OH SE SE SE L 2 0N 0 o1 1823 K 2-1 K(11 K2_1 C)C 2 HOH OH SE SE SE L 2 ON 5 6 0 1 1824 K 2-1 K11K(3-1 OC 2 HOH SE SE SE SE L 2 ON 5 6 01 1825 Ki- K1K 3- OC 2 HOH SE SE SE SE L 2 ON 560 1 1826 K 2 14_1'- OC 2
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4 OE SE SE SE SE L 2 ON 5 6 0 1 1827 K1(11 4_1 OC 2
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4 OE SE SE SE SE L 2 ON 5 6 01i 1828 K 2-1 K(1i1K(2-1 OH SE SE SE SE L 2 ON5_6 0 1 1829 K 21 I1 K3-1 0C 2
H
4 0H OH SE SE SE Li N6-6' 0 2 13 1_1 11i 3-1 CH0 OH SE SE SE Li O6-602 1830 K 1K OC 2
E
6 OH H S H HL ON6_ 0 2 1831 i- K(1(11 K4-1 OC 2
E
6 OE OH SE SE SE Li N6-6 0 2 1832 K2-1 OC 2
E
4 OE OH SE SE SE Li N6-6 0 2 184 K2-1 11 2 31 O2,H S SE L 1 O 6-602 1833 Ki- K1K3-_ OC 2
H
4 0H OE SE SE S ON6- 0 2 1834 (2_1 K(-11(4_1 OC 2
E
4 OE SE SE SE SE Li ON 6 6 0 2 1835 K(11 11 K4-1 OC 2
H
4 0H SE SE SE SE Li ON 6 6 0 2 1836 12-1 K1I<4 21 OCEOE SE SE SE SE L 1 ON r- 6 0 2 189 1e1 11- 1 CH0 H H S HL2 O 6-6o1 1837 K- K K3-' OC 2
E
4 OE SE SE SE SE L 2 ON" 021 14 211_121- C2 H O SE SE SE 2 O 6-6o1 1838 Ki- K K- OC 2 H40H SE S S S ON 026o 1843 K- K1_ 1 K2_1 OC 2 HOH OH SE SE SE L 2 ON 6 6 0 1 14 211111 OCH0 H SE SE SE 2 O 6-6 1 1840 Ki- OC 2 H4H SH S OE S ON 660 1 1841 leC 1(4-1 OC 2 HOH OH SE SE SE L 2 ON" 0 1 1842 K1_ 1(2-1 OC 2
H
4 OE OH SE SE SE L 2 ON 660 1 1843 (2_1 1_1 1(3_1 OC 2 H4OE OH SE SE SE Li ON 7 6 0 2 180 1i- K11 K31 2 6CH0 H S H S i O -602 1844 feC KlC e-1 OC 2
H
4 OH SE SE SE SE Li ON 760 2 1852 KiC K1_ K4 OC 2
E
4 OE SE SE SE SE Li ON -60 1 1849 fe21 K1_1 K2 1
OC
2
H
4 0H OH SE SE SE Li ON 7- 6 0 2 S:/ChemicaUSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 69 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 K2-1 K1-1 K3-1 K2-1 K1_1 K 4-1 K1-1 K_1 K 4 -1
K
2 -1 K1' K 2-1 K2-1 K'I' K 3 1 K1-1 K'1 K 3 -1 K2-1 K1-1 K 4 -1 K'1 K1_1 K 4 K2-1 K1_1 K 2 -1
K
2 -1 K_1 K 3 K''I K 3
K
2 -1 K1_1 K 4 1 K''l K 4 1-1 4-21 K K1_1 K 2-1 K1_1 K 2-1 K2-1 K1_1 K 3 -1_ K'1 K 3 -1
K
2 -1 K1_1 K 4 -1
K
4 K2-1 K1-1 K2-1 K2-1 K1- K 3-1 K 11 K11 1 3-1 K2-1 K1_1 K 4-1 K 4-1 K- K 2- K-1 K' K3-1 K1-' K 3-1 K2-' K11 K4-1 K1-' K' K 4-1 K2-1 K1_1 K 2-1 K2-' K1_1 K3-1 K'-1 K'1 K 3-1 K2-' K1_1 K4-1_ K11 K1_1 K 4-1 K2-' K1' K2-1 K2-1 K11 K 3-_ K''I K'1 K3-' K2-1 K1_1 K4-1 K' K1' K 4 -1
K
2 K1_1 K2-1 K-1 K-l K 3 -1_ OC2H 4 0H OC2H 4
OH
OC2H, 4
OH
OC2H 4
OR
OC2H 4
OH
OC2H 4
OH
OC2H 4
OH
OC2H 4
OR
OC2H 4
O
OC2H 4
O
OC2H 4
OR
OC2H 4 0H OC2HOH OC2H 4
O
OC2H40H OC2H 4
O
OC2H 4
OH
OC2H 4
OR
OC2H, 4
O
OC2H40H OC2H 4
OH
OC2H 4
OR
OC2H40H OC2H, 4
OH
OC2H40H OC2H40H OC2H 4 0I OC2H 4
OH
OC2H 4
OR
OC2H, 4
O
OC2H 4
OH
OC2H 4
OR
OC2H40H OC2H 4
O
OC2H 4 0H OC2H40H OC2H 4
OR
OC2H 4
OR
OC2H 4
OR
OC2H 4
OR
OC2H40H SH SH SR SR L 1 ON7- 6 02 SH SR SH SH SH SR SH SH OH SR OH SR OH SH OH SH OH SR SH SR SH SH SH SR SH SR SH SR OH SR OH SH OH SR OH SR OH SR SH SR SH SR SH SR SH SR SH SR OH SR OH SR OH SR OH SR OH SR SH SR SH SR SH SR SH SR SH SR OH SR OH SR OH SR OH SR OH SR SH SR
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SR
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
ON7-6 0 2 ON7- 6 0 2 ON7-6 0 2
ON
7 6 0 2 ON7- 6 0 1 N7-6 0 1
ON
7 6 0 1 0 1 7-6 ON- 0 1 ON7- 6 0 1 N7-6o
ON
7 6 0 1 ON7- 6 0 1 ON7- 6 0 1 ON7-6 0 2 ON-6 0 1
ON
9 6 0 2
ONS
6 0 2 ON_60 ON8 6 0 2 ON8 6 0 2 ON8-6 0 2
ON
9 6 0 2
ON
9 6 0 2
ON
9 6 0 2 ON 8-6 o i ON B-6 o 1 ON"_6 0 2 ON8 6 0 1 ON8-6 0 1 ON8 6 0 1
ON
9 6 0 1
ON
9 6 0 1 ON8 6 0 1
ON
9 6 0 1
ON
9 6 0 1 ON9-6 0 1
ON
9 6 0 1 0N 0 1
ON
9 6 0 2 ON9-6 0 2 S:/lChemical/Sankyo/FP200343/FP00343s .doc P89346/FP-200343/gds-mg/specification/03.05.05 70 1895 K''I CCHOH SE SE SH SE Li CN 9 6 0 2 1896 K 2 K1_ 1
OC
2 HOH SH SE SH SE Ll CN 9 6 0 2 1897 K''I C 2
H
4 CH SH SH SH SE Li ON 9 6 0 2 19 2_1 K1121- O2H SH SH SE SH Li N- 0 2 1898 K2_ OC 2 HOCH0S H HL N9- 1990 K 2 1
KI
1
K
3 1 OC 2 HOE CE SE SE SE L 2
CN
9 6 0 1 1900 K 1 1 I K'' 3 1 XCHOE OE SE SE SE L 2
ON
9 6 01i 1901 K 2 Kl 1
.OC
2 HOE OE SE SE SE L 2 N9-6 0 1 194 12- K11 CHO S S HL2 N9_601 1902 K- K K 3- C 2
E
4 OE CE SE SE SE L2 ON9_ 0 1 1903 K 2 1
K
1 K- C HOE CE S H SEH SE L 2
ON
9 6 0 1 1904 K 2 '1 K'- 1 2HEOE SE SE SE S H L 2 0 1 191 11- 21 C O SE SEH SEH SEL N9-6 1 1905 K2_ Ki K3- CC 2
H
4 0H O S H HL O0 0 1 1910 K 2 1
K-
1 OCHOH SE SE SE SE Li ON 9 01 0C2 1907 K2- 1 K'lK'- CC 2
E
4 OE SE SE SE SE L 2
ON
9 01 0C2 1922- K- 41 CH0 SE SE SE SE Li 9-6_10 1908 K_1 K_1 K2_ CC 2
E
4 0H O S HHL ON11 C 1 1909 K 2 1 K 3
OC
2
E
4 CE CE SE SE SE Li 0 2 1910 K 1 'i K''iK 3 1
OC
2
E
4 CE CE SE SE SE Li CN 1 1 0 2 1911 K 2
K
4 1 CC 2 HCE CE SE SE SE Li CN"'10 2 1912 K''1 K_1K_1 OC 2
E
4 OE CE SE SE SE Li CN 1 002 1913 K 2 K' 'K 2
CC
2 HE CE SE SEH SE Li CN' 1 0_02 1914 K 2 K' K 3 1
CC
2
E
4 0E SE SE SE SE L2 CN' 1 02 1915 K''i K' 1
K
3 1
C
2
E
4 OE SE SE SE SH L 2 CN0-Co2 1921 K2- K' K4-' CC 2
E
4 0E SE SE SE SE L2 C 2 1917 K'- 1 K_1 K4' CC 2
E
4 OH SE SE SE SE L 2 oO' 0 2 1918 K 2 1
K''K
2
CC
2
E
4 0E SE SEH SE SE L2 ON" 0 C 2 12 2_1 11- K3_1 CEH0H S SEH SE SE L 2 N10101 1919 Ki- KI K 3- CC 2
E
4 0H S S E- HL ON 01 0 1 196 121 11_ 341 CEH0H S SH SE SE L2 O10101 1920 K1_ K1_ K4- OC 2
H
4 CE S S H HL ON 01 C 1 1921 K 2 Kl 1 K2-' CHOEH CE SE SE SE L 2 ON0C11 1922 K''K2_ 1
OC
2
H
4 CE CE SE SE SE Li ON"' 01 1930 K 2
K'K
2 1 OCE0 CE SE SE SE Li CN2 1 012 1924 K 2 K2-' CC 2
H
4 0H SE OH SE SE Li o13-1 0 1 192 K2 1 K1_ 1 K2_ 1
CC
2
E
4 0H SE SE SE SE Li ON 1 4 C 1 1929 1(2-1 K 1 1
K
2
OC
2
H
4 0E OE CE CE CE L 1
ON
1 'C 02 1931 K 1 K''K2_ 1
CC
2
E
4 0E OH CE CE CE L 1
ON
1 6- 1 0 2 1935 K_1K2_1 0C 2 HOH OH CE OH OH L' ON 17 1 C 2 S:/ChemicaUSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 71 1936 K 2
K
1
K
2
OC
2
H
4 OH OH OH OH OH L 2
ON
14 -1 0 1 1937 K 2 -1 K 1
K
2 1
OC
2 H40H OH OH OH H 0 0 1938 K 2 1
K
1 1
K
2
OC
2
H
4 0H OH OH OH SC 2
H
4
C
18 0 H 0 1 1939 K 2 1
K
1 1
K
2 1
SC
2
H
4
C
2 0 OH SH SH H 0 0 1940 K 2 -1 K 1
K
2 -1 SC 2
H
4
C
18 OH SH SH H 0 0 1941 K 2 1
K'
1
K
2 1
SC
2 H40H OH SH SH H 0 0 1942 K 2 1
K
1
K
2
SC
2
H
4
C
20 SH SH SH H 0 0 1943 K2-' K-1 K 2 1 SC2H4C 8 SH SH SH H 0 0 1944 K 2 -1 K' K 2
SC
2
H
4 OH SH SH SH H 0 0 1945 K 2
K'
1
K
2
OC
2
H
4 0H OH SH SH SC 2
H
4 0H H 0 1 1946 K 2 1
K
1 1
K
2 1
OC
2
H
4 0H SH SH SH SC 2
H
4 OH H 0 1 In Table 1, Ph represents a phenyl group, Bn represents a benzyl group, Me represents a methyl group, Et represents an ethyl group, Pr represents an n-propyl group and tBu represents a tert-butyl group; and in Table 1, the groups described as K" represent groups having the following structure.
S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 72
NH
2
N
0Me 0 K2-1
NH
2 N
N
<K
0 N N 0 0 0
NH
2 Nr 0 N N 0 0 0
NH
2 0
N
K
3 4
NH
2 N
N
0 N N ;04 0 0
K
3 K 4-1 K4-2 K 43K4 Further, in Table 1, the groups described as Gly, POMO, 1 2 2 81 POMS, ATE, PTE, ALIM, L L c20, c8, c1 4 and C1 represent groups having the following structures respectively.
S:/ChemicaUSankyo/FP200343/FP200343s.doc S:/cenical~anko/F20043/F20043sdocP89346/FP-200343/gds-mg/specification//03.05.05
H
2
C-OH
HC-OH
-O-CH
2 Gly 0
H
3
C
H
3 CA 0 0-
H
3
C
POMO
0
H
3
C
H
3 CA
H
3
C
PTEO
HIC0
H
3
C
H
3 CA 0 S-
H
3
C
POMS
0 H C
H
3
H
3
C
PTES
0
ATES
0
H
3
CLS~~
ATEO
CH
3 1
OCH
3 H 0
ALM
-00CH 3 H
H
3 Cc2 0 -0 CH 3 c 18 0 -0 k- CH 3 C 14 0 -0 t CH 3 C 10 Further, in Table 1, the groups described as ONx represent oligonucleotide analogs having the structures defined P:\OPER\PDB\Speci\2003204578 1spa.doc 7/7/05 74 below and bonded to R 7 at the terminal.
N121 ON' -2 -G -ep G -ep G -ep G -ep A -ep C -ep A -e p-Ae-hp ON 1-4 -G--epG--epG--n S S S-A- S -An_-SAn_ S -An- S -A C -p A -eh ON' -G--epG--epGepG-S-' G- S-Gn- S-ePA-PA--e p-Ae-hp S-Ae-S-GeS-C -e h ON' -7 p-Ce-p -Ce-pAe Te- hp- ON 2-1 p-Te-hp S:/ChemicailSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 75 ON 2-3 p-Te-hp ON 2-6 S-Te-SCehp eSTeh ON 2- ON 3-1 -Ge-p-Te-p-Ae-p-Ce-p-Te-- Ce-pCe-pCe-p-e -p-Ge-p-Ce-p-T e -p-G Te pCe p-T Te-p-T--eGepe -hp
ON
3 -3 ON 3 S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/speciflcation/03.0505 76 p -Te-p-Ge -hp ON' 6 ON 3s-Te-s-Ge-hp ON 4-2 ON' -3 hp
ON
4 -4 p.-GT-s-Te- T pAe-p-e -C pA-h
ON
4 ON' S:/ChemicaIISankyoIFP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.0505 77 ON -l hP- e--e h ON ON N526
ON-
S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mgfspecification//03.05.05 78
ON
6 3 hp ON 6- S-Tn-S-C--e G-pG--eh ON S-nSC-SA--e S-e -G C- -T-eca h-ps~C~~e
ON
6 -7 ON 7-1 s -Ge s Ge- C-ep-hp ON 7-3 hp S:/Chemical/Sankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//03.05.05 79 T -p C e- -eh ON 7- -GeSC--esGeSA-- -AS-T n_A -s-G n_-n ON -7 -ep T- -ep A _nSGnSAnS- -e ON 8-1 ON e P Te -C C p-T e-.pG e .p..Ce -hp ON -C -p hp O N 8-4 n n ON' S:/ChemicallSankyo/FP200343/FP200343s.doc P89346/FP-200343/gds-mg/specification//O3.05.05 80 S-S 'e-S -C'--eSeS-hp ON8
ON'-
ON 9hp
ON
9 4 _T~_epC~_epG__n s-Cn_--Tn-S -Gn-S-Tn-S-Gn-S-An- S-Ca S-An- S-Tn-S- e- -ep A -T h
ON
9 S-TsS-T_-s _S-ASSGeepTeCeh ON 9- S-AeS- Te -S-Te-hp S:/ChemicaUSankyo/FP200343/FP200343s.doc S:/CemiaIIankoIF20033IF20043sdocP89346/FP-200343/gds-mg/specific-aioi/03.05.O5 81 hp ON11-1 AnpG-- e-G----epAehp ON14-1 An -p-Gn-p-Te-p- Te-~p-TepCe -pAe -hp 16 ON 1-1 -p A -ep G -ep C C _np T p T -np Further, the groups described as A n, G n. Tn, Ae, Ge, Ce, Te, p, s and hp in the above represent groups having the following structure.
S:/ChemicaUSankyo/FP200343/FP200343s.doc S:/cemicll~nkyoFP20343FP20343sdocP89346/FP-200343/gds-mg/specificaion//03.05.O5
NH
2
NN
0 0 N N 0 0 C
H
3 11NHX 0 N 1 0 I K N H 0 N 'NH 2 0
NH
2
H
3 C
N
I N"o 0
CH
3 NH 0 N 0 0 0 0 11
OH
p 0 11
SH
S
9P-/\/OH
OH
hp In the base sequences of the aforementioned oligonucleotide analogs, ON' is a sequence in human telomerase S:/Chemical/Sankyo/FP200343FP200343s1. .doc P89346/FP-200343/gds-mg/specificationl/03.05.05 (GenBank Accession No. U86046, base sequence of the complementary chain of nucleotide numbers 170 to 188), ON 2 is a sequence in human breakpoint cluster region (BCR) mRNA (GenBank e Accession No. NM-021574.1, base sequence of the complementary chain of nucleotide numbers 597 to 614), ON 3 is a sequence in 00 interferon-inducible double-stranded RNA-dependent human protein r- V) kinase (PKR) mRNA (GenBank Accession No. NM-002759.1, base 00 sequence of the complementary chain of nucleotide numbers 490 to Cc} 508), ON 4 is a sequence in human protein kinase C, alpha (PKCa) mRNA (GenBank Accession No. NM-002737.1, base sequence of the complementary chain of nucleotide numbers 2044 to 2063), ON 5 is a sequence in human intercellular adhesion molecule (ICAM1) mRNA (GenBank Accession No. NM-000201.1, base sequence of the complementary chain of nucleotide numbers 2100 to 2119), ON 6 is a sequence in human ras transforming protein gene (GenBank Accession No. M38453.1, base sequence of the complementary chain of nucleotide numbers 121 to 140), ON 7 is a sequence in human tumor necrosis factor (TNF superfamily, member 2) (TNF) mRNA (GenBank Accession No. NM-000594.1, base sequence of the complementary chain of nucleotide numbers 279 to 298), ON 8 is a sequence in human phosphotyrosyl-protein phosphatase (PTP-1B) mRNA (GenBank Accession No. M31724.1, base sequence of the complementary chain of nucleotide numbers 951 to 970), ON 9 is a sequence in human c-raf-1 mRNA (GenBank Accession No. NM- 002880.1, base sequence of the complementary chain of nucleotide numbers 2484 to 2503), and ON 10 is a sequence in human telomerase mRNA (GenBank Accession No. U86046, base sequence of the complementary chain of nucleotide numbers 136 to 148).
In the above Table 1, the preferred compounds are 1, 2, 3, 4, 5, 6, 7, 8, 13, 22, 27, 28, 31, 39, 41, 42, 50, 52, 53, 61, 63, 64, 71, 73, 77, 79, 96, 98, 102, 104, 146, 148, 152, 154, 171, 173, 177, 179, 290, 292, 293, 305, 307, 310, 311, 312, 313, 314, 316, 319, 320, 325, 330, 334, 338, 339, 343, 344, 351, 356, 364, 369, 377, 382, 386, 390, 391, 395, 396, 403, 408, 416, 421, 424, 425, 428, 438, 441, 451, 452, 453, 454, 455, 461, 462, 463, 464, 465, 471, 472, 473, 474, 475, 481, 482, 483, 484, 485, P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 491, 514, 542, 565, 593' 621, 644, 672, 695, 723, 751, 774, 802, 825, 853, 881, 907, 932, 961, 1015, 1078, 1099, 1146, 1207, 1345, 1356, 1449, 1490, 1531, 1572, 1621, 1653, 1694, 1723, 1754, 492, 515, 543, 571, 594, 622, 645, 673, 701, 724, 752, 775, 803, 831, 854, 882, 908, 936, 962, 493, 494, 495, 521, 522, 523, 544, 545, 551, 572, 573, 574, 595, 601, 602, 623, 624, 625, 651, 652, 653, 674, 675, 681, 702, 703, 704, 725, 731, 732, 753, 754, 755, 781, 782, 783, 804, 805, 811, 832, 833, 834, 855, 861, 862, 883, 884, 885, 909, 913, 914, 937, 941, 942, 963, 966, 967, 501, 524, 552, 575, 603, 631, 654, 682, 705, 733, 761, 784, 812, 835, 863, 891, 915, 943, 978, 1039, 1086, 1107, 1170, 1255, 1349, 1360, 1453, 1509, 1550, 1591, 1637, 1668, 1698, 1727, 1758, 502, 525, 553, 581, 604, 632, 655, 683, 711, 734, 762, 785, 813, 841, 864, 892, 919, 947, 979, 1050, 1087, 1110, 1171, 1267, 1350, 1361, 1469, 1510, 1551, 1592, 1641, 1669, 1705, 1728, 1774, 503, 504, 505, 511, 512, 513, 531, 532, 533, 534, 535, 541, 554, 555, 561, 562, 563, 564, 582, 583, 584, 585, 591, 592, 605, 611, 612, 613, 614, 615, 633, 634, 635, 641, 642, 643, 661, 662, 663, 664, 665, 671, 684, 685, 691, 692, 693, 694, 712, 713, 714, 715, 721, 722, 735, 741, 742, 743, 744, 745, 7.63, 764, 765, 771, 772, 773, 791, 792, 793, 794, 795, 801, 814, 815, 821, 822, 823, 824, 842, 843, 844, 845, 851, 852, 865, 871, 872, 873, 874, 875, 893, 894, 895, 901, 902, 903, 920, 924, 925, 926, 930, 931, 948, 949,953, 954, 959, 960, 990, 991, 1002, 1003, 1014, 1026, 1027, 1079, 1082, 1102, 1103, 1147, 1158, 1220, 1231, 1346, 1347, 1357, 1358, 1450, 1451, 1491, 1492, 1532, 1533, 1573, 1589, 1625, 1629, 1663, 1665, 1695, 1696, 1724, 1725, 1755, 1756, 1038, 1083, 1106, 1159, 1243, 1348, 1359, 1452, 1493, 1549, 1590, 1633, 1666, 1697, 1726, 1757, 1051, 1090, 1111, 1182, 1279, 1351, 1429, 1470, 1511, 1552, 1593, 1645, 1671, 1706, 1734, 1775, 1062, 1063, 1074, 1075, 1091, 1094, 1095, 1098, 1122, 1123, 1134, 1135, 1183, 1194, 1195, 1206, 1291, 1303, 1315, 1344, 1352, 1353,.1354, 1355, 1430, 1431, 1432, 1433, 1471, 1472, 1473, 1489, 1512, 1513, 1529, 1530, 1553, 1569, 1570, 1571, 1609, 1609, 1613, 1617, 1647, 1648, 1650, 1651, 1690, 1691, 1692, 1693, 1707, 1708, 1709, 1710, 1735, 1736, 1737, '1738, 1776, 1777, 1778 1794, 1795, 1796, 1797 1798, 1814, 1815, S:/Chemical/Sankyo/FP200343/FP200343s I .doc 1816, 1817, 1818, 1834, 1835, P89346/FP-200343/gds-mg/specification//03.05.05 1836, 1837 1838, 1854, 1855, 1856, 1857, 1858, 1874, 1875, 1876, 1877, 1878, 1894, 1895, 1896, and 1918, and more preferable 1897, 1898, 1914, 1915, 1916, 1917, 290, 455, 501, 542, 585, 631, 672, 715, 761, 802, 845, 305, 461, 502, 545, 591, 632, 675, 721, 762, 805, 851, 307, 462, 505, 551, 592, 635, 681, 722, 765, 811, 852, 338, 465, 511, 552, 595, 641, 682, 725, 771, 812, 855, 343, 471, 512, 555, 601, 642, 685, 731, 772, 815, 861, 364, 472, 515, 561, 602, 645, 691, 732, 775, 821, 862, compounds 369, 390, 475, 481, 521, 522, 562, 565, 605, 611, 651, 652, 692, 695, 735, 741, 781, 782, 822, 825, 865, 871, are 1, 2, 395, 416, 482, 485, 525, 531, 571, 572, 612, 615, 655, 661, 701, 702, 742, 745, 785, 791, 831, 832, 872, 875, 3, 4, 5, 8, 421, 451, 452, 491, 492, 495, 532, 535, 541, 575, 581, 582, 621, 622, 625, 662, 665, 671, 705, 711, 712, 751, 752, 755, 792, 795, 801, 835, 841, 842, 881, 882, 885, 891, 892, 895,953, 954, 959, 960, 961, 962, 963, 966, 967, 978, 979, 990, 991, 1002, 1003, 1014, 1015, 1026, 1027, 1038, 1039, 1050, 1051, 1062, 1063, 1075, 1079, 1083, 1087, 1091, 1095, 1099, 1103, 1107, 1110, 1111, 1122, 1123, 1134, 1135, 1146, 1147, 1158, 1159, 1170, 1171, 1182, 1183, 1194, 1195, 1206, 1207, 1429, 1430, 1449, 1450, 1469, 1470, 1489, 1490, 1509, 1510, 1529, 1530, 1549, 1550, 1569, 1570, 1589, 1590, 1648, 1650, 1651, 1653, 1666, 1668, 1669, 1671, 1691, 1692, 1695, 1696, 1697, 1698, 1707, 1708, 1709, 1710, 1725, 1726, 1727, and 1728.
[Mode for carrying out the invention] The compounds of the present invention can be prepared by appropriately utilizing Process A, Process B, Process C, Process D, Process E, Process F, Process G, and Process H mentioned below.
S :/ChemicalSankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mgspecification/03.05.05 Process A 0
II
H
A-2 (2) A-i RioP 3
)R
A-3 0
R
1
-OH
O-R
H-R
1 1 H (6) Process B B-1 0 9- 1 2 _R 11 11 -3 12_ 11 B-2 12 11 R P-OH -R R -R P\ 1 H R 1 (9)I B-4 0 12_ 11 1 R R -P-OH
O-R
Process C
R
1 20
B'
DO OH (11) R12 0B
R-
DO 0 R 9 0 1 (12) 1281 D-1 )-0j Process D (13) S:/ChemicaUSankyo/FP200343/FP200343s I.doc S:/Cerncal~anyo/P20043/P20343 1 docP89346/FP-200343/gds-mg/specification//03.05.05 Process E E-1 E-2
H-R
7
-R
8 -OH R 12
R
7
R
8 OH R 1 2
-R
7
-R
8 -O~R 13 OH (16) (1 0 E-3 0 0 E-4 0 R 12
R
7
R
8 0 R13 R1 H-R 15® (19) 0 0 Process F R12 0'
B
1) R 6 0 or (14) R 9O0N P1Rio (21) 2) or derivatives (1) Process G R12 0 Bi 00 im 2-5A derivatives (14) or (2 1)(1 or (22) Process H 0 0 0 derivatives (1) (14) or (2 1) In Process A, Process B, Process C, Process D, Process E, Process F, Process G, and Process H, A, D, and R' have the same meanings as defined above; R 9 represents a protecting group for protecting a phosphoric acid group or a phosphorous S:/Chemical/Sankyo/FP200343/FP200343s Ldoc S:/cemiall~nky/FP2034IFP2034s 1 docP89346/FP-200343/gds-mgspecification/03.O5.05 88 acid group; R" represents a dialkylamino group (particularly a diisopropylamino group or a diethylamino group); R 11 represents an R 1 group which requires a protecting group in the synthesis C of the 2-5A analog; B 1 represents a purin-9-yl group or a substituted purin-9-yl group having substituent(s) selected from 00 the above Group a, but a group substituted by amino group is tf3 excluded. R 12 and R 16 are the same or different and represent a 00 protecting group; R" represents a -(CH 2 group (h is an o integer of from 2 to R 14 represents a hydroxyl group, a Sphenyloxy group which may be substituted, or an ethyloxy group which may be substituted by halogen; R 15 represents an oxygen atom, a sulfur atom or an NH group; and HR 15 -P (encircled) represents a high molecular weight compound.
The "protecting group" in the definition of R 9 can be, for example, a lower alkyl group such as methyl; a lower alkenyl group such as 2-propenyl; a cyano lower alkyl group such as 2cyanoethyl; a lower alkoxylated lower alkoxymethyl group such as 2-methoxyethoxymethyl; a halogeno lower alkoxymethyl group such as 2,2,2-trichloroethoxymethyl and bis(2-chloroethoxy)methyl; a halogenated ethyl group such as 2,2,2-trichloroethyl; a methyl group substituted by an aryl group such as benzyl; a methyl group substituted by from 1 to 3 aryl groups whose aryl ring is substituted by lower alkyl, lower alkoxy, halogen or cyano group(s) such as 4-methylbenzyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl and 4-cyanobenzyl; an aryl group substituted by halogen atom(s), lower alkoxy group(s) or nitro group(s) such as 4-chlorophenyl, 2-chlorophenyl, 4methoxyphenyl, 4-nitrophenyl and 2,4-dinitrophenyl; or a lower alkylcarbonyloxymethyl group such as pentanoyloxymethyl and pivaloyloxymethyl; and is preferably a methyl group, a 2cyanoethyl group, a benzyl group, a 2-chlorophenyl group, a 4chlorophenyl group, a 2-propenyl group or a pivaloyloxymethyl group.
The "protecting group" in the definition of R 12 and R 16 can be, for example, an "acyl type" protecting group including an "aliphatic acyl group" such as an alkylcarbonyl group, e.g., P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1methylpentadecanoyl, 14-methylpentadecanoyl, 13, 13dimethyltetradecanoyl, heptadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl and heneicosanoyl; a carboxylated alkylcarbonyl group, e.g., succinoyl, glutaroyl and adipoyl; a halogeno lower alkylcarbonyl group, chioroacetyl, dichioroacetyl, trichioroacetyl and trifluoroacetyl; a lower alkoxy lower alkylcarbonyl group, e.g., methoxyacetyl; or an unsaturated alkylcarbonyl group, 2-methyl-2-butenoyl; and an "aromatic acyl group" such as an arylcarbonyl group, benzoyl, ax-naphthoyl and f-naphthoyl; a halogeno arylcarbonyl group, 2-bromobenzoyl and 4-chlorobenzoyl; a lower alkylated arylcarbonyl group, 2,4,6-trimethylbenzoyl and 4-toluoyl; a lower alkoxylated arylcarbonyl group, 4anisoyl; a carboxylated arylcarbonyl group, 2carboxybenzoyl, 3-carboxybenzoyl and 4-carboxybenzoyl; a nitrated arylcarbonyl group, 4-nitrobenzoyl and 2nitrobenzoyl; a lower alkoxycarbonylated arylcarbonyl group, 2-(methoxycarbonyl)benzoyl; or an arylated arylcarbonyl group, 4-phenylbenzoyl; a "lower alkyl group" such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2methylbutyl, neopentyl, l-ethylpropyl, n-hexyl, isohexyl, 4methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3, 3-dimethylbutyl, 2, 2-dimethylbutyl, 1, 1-dimethylbutyl, 1,2dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl and 2ethylbutyl; a "lower alkenyl group" such as ethenyl, 1-propenyl, 2-propenyl, l-methyl-2-propenyl, l-methyl-l-propenyl, 2-methyl-l-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, l-methyl-l-butenyl, 3-methyl-2-butenyl, 1- S:/Chemical/Sankyo/FP200343IFP200343s1. .doc P89346/FP-200343/gds-mg/specification//03.05.05 ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3butenyl, l-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl, 1-methyl-2pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-pentenyl, 2-methyl- 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 00 hexenyl; a "tetrahydropyranyl or tetrahydrothiopyranyl group" such as 00 tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4methoxytetrahydropyran-4-yl, tetrahydrothiopyran-2-yl and 4methoxytetrahydrothiopyran-4-yl; a "tetrahydrofuranyl or tetrahydrothiofuranyl group" such as tetrahydrofuran-2-yl and tetrahydrothiofuran-2-yl; a "silyl group" such as a tni-lower alkylsilyl group, e.g., trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, tbutyldimethylsilyl, methyldiisopropylsilyl, methyldi-tbutylsilyl and triisopropylsilyl; or a tni-lower alkylsilyl group substituted by 1 or 2 aryl groups, e.g., diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl and phenyldiisopropylsilyl; a "lower alkoxymethyl group" such as methoxymethyl, 1,1dimethyl-1-niethoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl and t-butoxymethyl; a "lower alkoxylated lower alkoxymethyl group" such as 2methoxyethoxymethyl; a "halogeno lower alkoxymethyl" such as 2,2,2- P:\OPER\PDB\Speci\2003284578 1spa.doc 7/7/05 trichloroethoxymethyl and bis(2-chloroethoxy)methyl; a "lower alkoxylated ethyl group" such as 1-ethoxyethyl and 1- (isopropoxy)ethyl; a "halogenated ethyl group" such as 2,2,2-trichloroethyl; a "methyl group substituted by from 1 to 3 aryl groups" such as benzyl, a-naphthylmethyl, 0-naphthylmethyl, diphenylmethyl, triphenylmethyl, a-naphthyldiphenylmethyl and 9-anthrylmethyl; a "methyl group substituted by from 1 to 3 aryl groups whose aryl ring is substituted by lower alkyl, lower alkoxy, halogen or cyano group(s)" such as 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl and 4cyanobenzyl; a "lower alkoxycarbonyl group" such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl; an "aryl group substituted by halogen atom(s), lower alkoxy group(s) or nitro group(s)" such as 4-chlorophenyl, 2chlorophenyl, 4-methoxyphenyl, 4-nitrophenyl and 2,4dinitrophenyl; a "lower alkoxycarbonyl group substituted by halogen or trilower alkylsilyl group(s)" such as 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl; an "alkenyloxycarbonyl group" such as vinyloxycarbonyl and aryloxycarbonyl; or an "aralkyloxycarbonyl group whose aryl ring may be substituted by 1 or 2 lower alkoxy or nitro groups" such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl and 4nitrobenzyloxycarbonyl.
In the following, the respective steps of Process A, Process B, Process C, Process D, Process E, Process F, Process G, and Process H will be explained in detail.
(Step A-i) The present step is a step, wherein compound is S:/ChemicalSankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 produced by reacting compound with a mono-substituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, monosubstituted chloro(benzyloxy)phosphine, or di-substituted benzyloxyphosphine normally used for amidite formation, in an inert solvent.
The solvent to be used is not particularly limited so long as it does not affect the reaction, but can preferably be an ether such as tetrahydrofuran, diethyl ether or dioxane; or a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene.
The mono-substituted chloro(alkoxy)phosphine to be used can be, for example, a phosphine such as chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine or chloro(diisopropylamino)cyanoethoxyphosphine, and is preferably chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine or chloro(diisopropylamino)cyanoethoxyphosphine.
In the case of using a mono-substituted chloro(alkoxy)phosphine, a deoxidizer is used. In that case, the deoxidizer to be used can be a heterocyclic amine such as pyridine or dimethylaminopyridine; or an aliphatic amine such as trimethylamine, triethylamine, diisopropylamine or diisopropylethylamine, and is preferably an aliphatic amine (particularly diisopropylethylamine).
The di-substituted alkoxyphosphine to be used can be, for example, a phosphine such as bis(diisopropylamino)cyanoethoxyphosphine, bis(diethylamino)methanesulfonylethoxyphosphine, bis(diisopropylamino)(2,2,2-trichloroethoxy)phosphine or bis(diisopropylamino)(4-chlorophenylmethoxy)phosphine, and is S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 preferably bis(diisopropylamino)cyanoethoxyphosphine.
In the case of using a di-substituted alkoxyphosphine, an acid or an organic salt is used. In that case, the acid to be used is tetrazol, acetic acid or p-toluenesulfonic acid, and the organic salt to be used is tetrazol diisopropylamine salt, acetic acid diisopropylamine salt or p-toluenesulfonic acid diisopropylamine salt, preferably tetrazol or tetrazol diisopropylamine salt.
The mono-substituted chloro(benzyloxy)phosphine to be used can be, for example, a phosphine such as chloro(morpholino)benzyloxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)benzyloxyphosphine or chloro(diisopropylamino)benzyloxyphosphine, and is preferably chloro(diisopropylamino)benzyloxyphosphine.
In the case of using a mono-substituted chloro(benzyloxy)phosphine, a deoxidizer is used. In that case, the deoxidizer to be used can be a heterocyclic amine such as pyridine or dimethylaminopyridine; or an aliphatic amine such as trimethylamine, triethylamine, diisopropylamine or diisopropylethylamine, and is preferably an aliphatic amine (particularly diisopropylethylamine).
The di-substituted benzyloxyphosphine to be used can be, for example, a phosphine such as bis(diisopropylamino)benzyloxyphosphine or bis(diethylamino)benzyloxyphosphine, and is preferably bis(diisopropylamino)benzyloxyphosphine.
In the case of using a di-substituted benzyloxyphosphine, an acid or an organic salt is used. In that case, the acid to be used is tetrazol, acetic acid or p-toluenesulfonic acid, and the organic salt to be used is tetrazol diisopropylamine salt, acetic acid diisopropylamine salt or p-toluenesulfonic acid diisopropylamine salt, preferably tetrazol or tetrazol diisopropylamine salt.
The reaction temperature is not particularly limited, but is normally from 0 to 80 0 C, preferably room temperature.
S:/Chemica/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 While the reaction time varies depending on the starting materials, the reagents and the temperature used, it is normally from 5 minutes to 30 hours; and in the case where the reaction is carried out at room temperature, it is preferably from minutes to 10 hours.
After the reaction, the desired compound of the present reaction is obtained, for example, by, after suitably neutralizing the reaction mixture, and removing any insoluble matter, if present, by filtration, addition of water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent. The thus obtained desired compound can be further purified by ordinary methods such as recrystallization, reprecipitation or chromatography, if necessary.
(Step A-2) The present step is a step, wherein compound is produced by allowing compound to react with tris-(1,2,4triazolyl)phosphite in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), and adding water thereto to cause H-phosphonation.
The reaction temperature is not particularly limited, but is normally from -20 to 100 0 C, preferably from 10 to 40 0
C.
While the reaction time varies depending on the starting materials, the reagents and the temperature used, it is normally from 5 minutes to 30 hours; and in the case where the reaction is carried out at room temperature, it is preferably 30 minutes.
After the reaction, the desired compound of the present reaction is obtained, for example, by, after suitably neutralizing the reaction mixture, and removing any insoluble matter, if present, by filtration, addition of water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the S:/Chemical/Sankyo/FP200343/FP200343s 1 .doc P89346/FP-200343/gds-mg/specification//03.05.05 like, and distilling off the solvent. The thus obtained desired compound can be further purified by ordinary methods such as recrystallization, reprecipitation or chromatography, if necessary.
(step A-3) The present step is a step, wherein compound is produced by allowing compound to react with a bis(1,2,4triazolyl)arylphosphate, bis(1,2,4-triazolyl)benzylphosphate, bis(1,2,4-triazolyl)-2-cyanoethylphosphate, bis(1,2,4triazolyl)(2,2,2-trichloroethyl)phosphate or bis(1,2,4triazolyl)(2-propenyl)phosphate in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), and adding water thereto to make a phosphodiester.
The bis(1,2,4-triazolyl)arylphosphate to be used can be, for example, bis(1,2,4-triazolyl)phenylphosphate, bis(1,2,4- 'triazolyl)(2-chlorophenyl)phosphate, bis(1,2,4-triazolyl)(4chlorophenyl)phosphate, bis(1,2,4-triazolyl)(2nitrophenyl)phosphate or bis(1,2,4-triazolyl)(4nitrophenyl)phosphate, and is preferably bis(l,2,4-triazolyl)(2chlorophenyl)phosphate or bis(1,2,4-triazolyl)(4chlorophenyl)phosphate.
The reaction temperature is not particularly limited, but is normally from -20 to 100 0 C, preferably from 10 to 40 0
C.
While the reaction time varies depending on the starting materials, the reagents and the temperature used, it is normally from 5 minutes to 30 hours; and in the case where the reaction is carried out at room temperature, it is preferably 30 minutes.
After the reaction, the desired compound of the present reaction is obtained, for example, by, after suitably neutralizing the reaction mixture, and removing any insoluble matter, if present, by filtration, addition of water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent. The thus obtained desired S:/Chemical/Sankyo/FP200343/FP200343s I.doc P89346/FP-200343/gds-mg/specification//03.05.05 compound can be further purified by ordinary methods such as recrystallization, reprecipitation or chromatography, if necessary.
(step B-l) 00 The present step is a step, wherein compound is produced by allowing compound to react with a protecting 00 reagent in the presence of a basic catalyst in an inert solvent.
C The solvent to be used can preferably be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated CN hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate or diethyl carbonate; an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane or diethylene glycol dimethyl ether; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonitrile or isobutyronitrile; an amide such as formamide, dimethylformamide (DMF), dimethylacetamide or hexamethylphosphortriamide; a sulfoxide such as dimethyl sulfoxide or sulfolane; an aliphatic tertiary amine such as trimethylamine, triethylamine or Nmethylmorpholine; or an aromatic amine such as pyridine or picoline; and is more preferably a halogenated hydrocarbon (particularly methylene chloride) or an aromatic amine (particularly pyridine).
The protecting reagent to be used is not particularly limited so long as it is adapted for the following nucleic acid synthesis and can be removed under acidic or neutral conditions, and can preferably be a tri-arylmethyl halide such as trityl chloride, mono-methoxytrityl chloride or dimethoxytrityl chloride; or a triarylmethanol ether such as dimethoxytrityl-0triflate.
In the case of using a tri-arylmethyl halide as the P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 protecting reagent, a base is normally used. In that case, the base to be used can be a heterocyclic amine such as pyridine, dimethylaminopyridine or pyrrolidinopyridine; or an aliphatic tertiary amine such as trimethylamine or triethylamine; and is preferably pyridine, dimethylaminopyridine or pyrrolidinopyridine.
In the case of using a liquid base as the solvent, since the base itself functions as a deoxidizer, it is not necessary to add a further base.
The reaction temperature varies depending on the starting materials, the reagents and the solvent used, and is normally from 0 to 150 0 C, preferably from 20 to 100 0 C. While the reaction time varies depending on the starting materials, the solvent and the reaction temperature used, it is normally from 1 to 100 hours, preferably from 2 to 24 hours.
After the reaction, the desired compound of the present reaction is obtained, for example, by concentrating the reaction mixture, adding water and an immiscible organic solvent such as ethyl acetate, followed by washing with water, separating the organic layer containing the desired compound, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent.
The resulting compound can be further purified by ordinary methods, for example, recrystallization or silica gel column chromatography, if necessary.
(Step B-2) The present step is a step, wherein compound is produced by allowing compound prepared in Step B-1 to react with a mono-substituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, mono-substituted chloro(benzyloxy)phosphine or di-substituted benzyloxyphosphine, which is normally used for amidite formation, in an inert solvent.
The present step is carried out similarly to Step (Step B-3) S:/Chemical/Sankyo/FP200343/FP200343sl.doc P89346/FP-200343/gds-mg/specification//03.05.05 The present step is a step, wherein compound is produced by allowing compound prepared in Step B-1 to react with tris-(l,2,4-triazolyl)phosphite in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), followed by adding water to carry out H-phosphonation.
The present step is carried out similarly to Step (Step B-4) The present step is a step, wherein compound is produced by allowing compound prepared in Step B-1 to react with a bis(1,2,4-triazolyl)arylphosphate, bis(1,2,4triazolyl)benzylphosphate, bis(1,2,4-triazolyl)-2cyanoethylphosphate, bis(1,2,4-triazolyl)(2,2,2trichloroethyl)phosphate, or bis(1,2,4-triazolyl)(2propenyl)phosphate in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), followed by adding water to make a phosphodiester.
The present step is carried out similarly to Step A-3.
(Step C-l) The present step is a step, wherein compound (12) is produced by allowing compound (11) to react with a monosubstituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, mono-substituted chloro(benzyloxy)phosphine, or di-substituted benzyloxyphosphine normally used for amidite formation, in an inert solvent.
Compound (11) is a compound wherein a nucleoside has been reacted with an alkyl halide such as methyl iodide or an alkenyl halide such as allyl bromide in the presence of sodium hydride, according to the method described in PCT/US94/10131, to obtain the 3'-substituted compound, and then the group, and amino group of the base portion, have been protected by protecting groups. For example, 3'-O-allyladenosine (catalogue No.: RP-3101) can be purchased from ChemGene Industries, and 5'-O-dimethoxytrityl-3'-O-allyl-Nbenzoyladenosine can be obtained therefrom by protection using S:/Chemical/Sankyo/FP200343/FP200343s I .doc P89346/FP-200343/gds-mg/specification//03.05.05 publicly known methods.
The present step is carried out similarly to Step A-i.
Amongst compounds 5'-O-dimethoxytrityl-3'-0- ^C methyl-N-benzoyladenosine-2'-0-(2-cyanoethyl N,Ndiisopropylphosphoramidite)(catalogue No.: ANP-2901), for 00 example, can be purchased from ChemGene Industries.
00 (Step D-l) C The present step is a step, wherein compound (14) is O produced by allowing compound (13) to react with a mono- (1 substituted chloro(alkoxy)phosphine, di-substituted alkoxyphosphine, mono-substituted chloro(benzyloxy)phosphine, or di-substituted benzyloxyphosphine normally used for amidite formation, in an inert solvent.
Compound (13) is the same compound as compound described in Process F of Japanese Patent Application (Kokai) No. 2002-249497, or the compound described in Japanese Patent Application (Kokai) No. Hei 10-195098 in which Yi is a protecting group and Y 2 is a hydrogen atom.
The present step is carried out similarly to Step (Step E-1) The present step is a step, wherein compound (16) is produced by allowing compound (15) to react with a protecting reagent in the presence of a basic catalyst in an inert solvent.
The present step is carried out similarly to Step (Step E-2) The present step is a step, wherein compound (17) is produced by allowing compound (16) prepared in Step E-1 to react with a dicarboxylic anhydride in an inert solvent.
The solvent to be used is not particularly limited so long as it does not inhibit the reaction and dissolves the starting material to a certain extent, and can be, for example, an aromatic hydrocarbon such as benzene, toluene or xylene; a P:\OPER\PDB\Speci\2003284578 Ispadoc 7/7/05 halogenated hydrocarbon such as methylene chloride or chloroform; an ether such as ether, tetrahydrofuran, dioxane or dimethoxyethane; an amide such as dimethylformamide, dimethylacetamide or hexamethylphosphortriamide; a sulfoxide such as dimethyl sulfoxide; a ketone such as acetone or methyl ethyl ketone; a heterocyclic amine such as pyridine; or a nitrile such as acetonitrile; and is preferably a halogenated hydrocarbon such as methylene chloride.
The deoxidizer to be used can be a pyridine such as pyridine, dimethylaminopyridine or pyrrolidinopyridine, and is preferably dimethylaminopyridine.
The dicarboxylic anhydride to be used is not limited so long as it is the anhydride of an a,o-alkyl dicarboxylic acid having from 3 to 16 carbon atoms, and can preferably be succinic anhydride.
While the reaction temperature and the reaction time vary depending on the acid anhydride and deoxidizer used, in the case where succinic anhydride is used, and dimethylaminopyridine is used as the deoxidizer, the reaction is carried out at room temperature for 30 minutes.
After the reaction, the desired compound is collected from the reaction mixture according to ordinary methods. For example, after suitably neutralizing the reaction mixture and removing any insoluble matter, if present, by filtration, water and an immiscible organic solvent such as ethyl acetate are added, followed by washing with water, separating the organic layer containing the desired compound, drying the extract with anhydrous magnesium sulfate or the like, and distilling off the solvent to obtain the desired compound. The resulting desired compound can be further purified by ordinary methods, for example, recrystallization, reprecipitation or chromatography if necessary.
(Step E-3) The present step is a step, wherein active ester (18) is formed by reaction of the carboxyl group of compound (17) having S:/Chemica/Sankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 a free carboxyl group with an ester-forming reagent in an inert solvent, and then reaction with a phenol which may be substituted.
The solvent to be used is not particularly limited so long as it does not inhibit the reaction, and it can be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate or diethyl carbonate; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonitrile or isobutyronitrile; an amide such as formamide, dimethylformamide (DMF), dimethylacetamide or hexamethylphosphortriamide; or a sulfoxide such as dimethyl sulfoxide or sulfolane; and is preferably a halogenated hydrocarbon (particularly methylene chloride) or an amide (particularly dimethylformamide).
The phenol to be used is not particularly limited so long as it can be used as an active ester, and it can be 4nitrophenol, 2,4-dinitrophenol, 2,4,5-trichlorophenol, 2,3,4,5,6-pentachlorophenol or 2,3,5,6-tetrafluorophenol, and is preferably pentachlorophenol.
The ester-forming reagent to be used can be, for example, an N-hydroxy compound such as N-hydroxysuccinimide, 1hydroxybenzotriazole or N-hydroxy-5-norbornene-2,3dicarboxyimide; a diimidazole compound such as 1,1'oxalyldiimidazole or N,N'-carbonyldiimidazole; a disulfide compound such as 2,2'-dipyridyldisulfide; a succinic acid compound such as N,N'-disuccinimidylcarbonate; a phosphinic chloride compound such as N,N'-bis(2-oxo-3oxazolidinyl)phosphinic chloride; an oxalate compound such as N,N'-disuccinimidyloxalate (DSO), N,N-diphthalimidyloxalate (DPO), N,N'-bis(norbornenylsuccinimidyl)oxalate (BNO), 1,1'bis(benzotriazolyl)oxalate (BBTO), 1,1'-bis(6chlorobenzotriazolyl)oxalate (BCTO) or 1,1'-bis(6- S:/ChemicalSankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 trifluoromethylbenzotriazolyl)oxalate (BTBO); or a carbodiimide such as dicyclohexylcarbodiimide (DCC); and is preferably a diimidazole compound or a carbodiimide (particularly DCC).
While the reaction temperature and the reaction time vary depending on the ester-forming reagent and the kind of the solvent used, the reaction is carried out at from 0°C to 100 0
C
for from 5 to 50 hours and, particularly in the case where pentachlorophenol and DCC are used in DMF, the reaction is carried out at room temperature for 18 hours.
After the reaction, the desired compound is collected from the reaction mixture according to ordinary methods. For example, after suitably neutralizing the reaction mixture and removing any insoluble matter, if present, by filtration, water and an immiscible organic solvent such as ethyl acetate are added, followed by washing with water, separating the organic layer containing the desired compound, drying the extract with anhydrous magnesium sulfate or the like, and distilling off the solvent to obtain the desired compound. The resulting desired compound can be further purified by ordinary methods, for example, recrystallization, reprecipitation or chromatography if necessary.
(Step E-4) The present step is a step, wherein high molecular weight derivative which can be used as a carrier for oligonucleotide synthesis, is produced by allowing compound (18) having an activated carboxyl group obtained in Step E-3 to react with a high molecular weight substance such as a control pore glass (CPG) bonded to an amino group, a hydroxyl group, a sulfhydryl group or the like through an alkylene group, in an inert solvent.
The high molecular weight substance (19) used in the present step is not particularly limited so long as it is used as a carrier, but it is necessary to examine the particle size of the carrier, the size of surface area by a three-dimensional network structure, the ratio of hydrophilic group positions, the S:/Chemical/Sankyo/FP200343/FP200343s1.doc P89346/FP-200343/gds-mg/specification//03.05.05 chemical composition, strength against pressure, and the like.
The carrier to be used can be a polysaccharide derivative such as cellulose, dextran or agarose; a synthetic polymer such as polyacrylamide gel, polystyrene resin or polyethylene glycol; or an inorganic substance such as silica gel, porous glass or a metal oxide. Specifically, it can be a commercially available carrier such as aminopropyl-CPG, long chain aminoalkyl-CPG (these are manufactured by CPG Inc.), Cosmoseal NH 2 Cosmoseal Diol (these are manufactured by Nacalai Tesque), CPC-Silica Carrier Silane Coated, aminopropyl-CPG-550A, aminopropyl-CPG-1400A, polyethylene glycol 5000 monomethyl ether (these are manufactured by Furuka Inc.), p-alkoxybenzyl alcohol resin, aminomethyl resin, hydroxymethyl resin (these are manufactured by Kokusan Kagaku Inc.) and polyethylene glycol 14000 monomethyl ether (these are manufactured by Union Carbide Inc.), but it is not limited to these.
Further, the functional group bonded to the carrier can preferably be an amino group, a sulfhydryl group, or a hydroxyl group.
The solvent used in the present step is not particularly limited so long as it does not inhibit the reaction and dissolves the starting material to a certain extent, and it can preferably be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate or diethyl carbonate; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonitrile or isobutyronitrile; an amide such as formamide, dimethylformamide (DMF), dimethylacetamide or hexamethylphosphortriamide; or a sulfoxide such as dimethyl sulfoxide or sulfolane; and is preferably a halogenated hydrocarbon (particularly methylene chloride), or an amide (particularly dimethylformamide).
The reaction temperature is normally from -20 to 150 0
C,
S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 preferably from 0 to 50 0 C. The reaction time varies depending on the starting materials, the solvent, and the reaction temperature used, but it is normally from 1 to 200 hours, preferably from 24 to 100 hours. After the reaction, the desired compound is collected from the reaction mixture according to ordinary methods. For example, the desired compound is obtained by recovering the high molecular weight carrier from the reaction mixture by filtration, washing with an organic solvent such as methylene chloride, and drying under reduced pressure.
(Step F-l) The present step is a step, wherein 2-5A analog is produced on a DNA automatic synthesizer by ordinary methods using the CPG (20) prepared in Step E-4, using the compounds (12) and (14) prepared in Step A-1, B-2, C-l or D-l, and a commercially available phosphoramidite reagent (21).
The 2-5A analog having the desired nucleotide sequence can be synthesized according to a method described in the literature (Nucleic Acids Research, 12, 4539 (1984)), and the manual attached to the synthesizer, by a phosphoramidite method using a DNA synthesizer, for example, model 392 of Perkin Elmer Inc.
As the compound for example, 3'-O-(t-butyldimethylsilyl)-N-benzoyladenosine-2'-0-(2cyanoethyl N,N-diisopropylphosphoramidite) can be purchased from ChemGene Inc. (catalogue No.: ANP-5681).
In the present step, the amidite reagent for the compounds (14) and (21) is activated using an acid catalyst to form a phosphorous acid tri-ester bond, and it is oxidized to a phosphoric acid tri-ester using an appropriate oxidizing agent, or it is made into a thiophosphoric tri-ester using an appropriate thioating agent.
The acidic substance used as a catalyst in the condensation reaction of the present step can be an acidic S:/Chemica/Sankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 105 substance such as a tetrazole, and is preferably tetrazole or ethylthiotetrazole. The oxidizing agent used in the oxidation reaction of the present step is not particularly limited so long as it is normally used in oxidation reactions, and is preferably an inorganic metal oxidizing agent such as a manganese oxide, 00 potassium permanganate or manganese dioxide; a ruthenium iq oxide, ruthenium tetraoxide; a selenium compound, i.e., 00 selenium dioxide; an iron compound, iron chloride; an osmium compound, osmium tetraoxide; a silver compound, o silver oxide; a mercury compound, mercury acetate; a (N lead oxide compound, lead oxide or lead tetraoxide; a chromic acid compound, potassium chromate, a chromic acidsulfuric acid complex, or a chromic acid-pyridine complex; or a cerium compound, cerium ammonium nitrate (CAN); an inorganic oxidizing agent such as a halogen molecule, a chlorine molecule, a bromine molecule or an iodine molecule; a periodic acid, sodium periodate; ozone; hydrogen peroxide; a nitrous acid compound, nitrous acid; a chlorous acid compound, potassium chlorite or sodium chlorite; or a persulfuric acid compound, potassium persulfate or sodium persulfate; or an organic oxidizing agent such as a reagent used in DMSO oxidation (a complex of dimethyl sulfoxide with dicyclohexylcarbodiimide, oxalyl chloride, acetic anhydride or phosphorus pentaoxide, or a complex of pyridine-sulfur trioxide); a peroxide such as t-butyl hydroperoxide; a stable cation such as triphenylmethyl cation; a succinic acid imide such as N-bromosuccinic acid imide; a hypochlorous acid compound such as t-butyl hypochlorite; an azodicarboxylic acid compound such as azodicarboxylic acid ester; a disulfide such as dimethyl disulfide, diphenyl disulfide, or dipyridyl disulfide and triphenylphosphine; a nitrous acid ester such as methyl nitrite; a carbon tetrahalide, carbon tetrabromide; or a quinone compound, 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ); preferably iodine.
The solvent to be used is not particularly limited so P:\OPER\PDB\Speci\2003284578 Ispadoc 7/7/05 long as it does not inhibit the reaction and dissolves the starting material to a certain extent, and it can preferably be an aromatic hydrocarbon such as benzene, toluene or xylene; a halogenated hydrocarbon such as methylene chloride or chloroform; an ether such as ether, tetrahydrofuran, dioxane or dimethoxyethane; an amide such as dimethylformamide, dimethylacetamide or hexamethylphosphortriamide; a sulfoxide such as dimethyl sulfoxide; an alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol or isoamyl alcohol; a dilute acid such as aqueous sulfuric acid; a dilute base such as aqueous sodium hydroxide; water; a ketone such as acetone or methyl ethyl ketone; a heterocyclic amine such as pyridine; or a nitrile such as acetonitrile; preferably a heterocyclic amine (particularly pyridine), a nitrile (particularly acetonitrile), an ether (particularly tetrahydrofuran), or a halogenated hydrocarbon (particularly methylene chloride).
Further, in the case where the compound is thioated, if desired, the thioate derivative can be obtained according to a method described in the literature (Tetrahedron Letters, 32, 3005 (1991), J. Am Chem. Soc., 112, 1253 (1990)) using a reagent such as sulphur, tetraethyl thiuram disulfide (TETD, Applied Biosystems Inc., or Beaucage reagent (Millipore Inc.) for forming a thioate by reacting with a phosphite.
The reaction temperature is normally from 0 to 150 0
C,
preferably from 10 to 60 0 C. The reaction time varies depending on the starting materials, the solvent and the reaction temperature used, but it is normally from 1 minute to 20 hours, preferably from 1 minute to 1 hour.
In the case where the H-phosphonic acid compound or obtained in Step A-2 or B-3 is condensed to form a phosphoric tri-ester bond in the present step, after it is condensed, for example, in the presence of a condensing agent S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 such as pivaloyl chloride and a deoxidizer to form the Hphosphonic acid diester bond, the H-phosphonic acid bond can be converted to the phosphoric acid diester bond using an oxidizing (y agent.
The solvent used in the present step is not particularly 00 limited so long as it does not inhibit the reaction, but l~ anhydrous acetonitrile is preferably used. As the reagent used 00 as the condensing agent, an acid chloride of a carboxylic acid Sor phosphoric acid is used, and pivaloyl chloride is preferably O used.
The oxidizing agent for oxidizing the ODN having a Hphosphonic acid bond to a phosphodiester type ODN is not particularly limited so long as it is normally used for oxidation reactions, and can be a inorganic metal oxidizing agent such as a manganese oxide, potassium permanganate or manganese dioxide; a ruthenium oxide, ruthenium tetraoxide; a selenium compound, selenium dioxide; an iron compound, iron chloride; an osmium compound, osmium tetraoxide; a silver compound, silver oxide; a mercury compound, mercury acetate; a lead oxide compound, e.g., lead oxide or lead tetraoxide; a chromic acid compound, e.g., potassium chromate, a chromic acid-sulfuric acid complex or a chromic acid-pyridine complex; or a cerium compound, e.g., cerium ammonium nitrate (CAN); an inorganic oxidizing agent such as a halogen molecule, a chlorine molecule, a bromine molecule or an iodine molecule; a periodic acid, sodium periodate; ozone; hydrogen peroxide; a nitrous acid compound, nitrous acid; a chlorous acid compound potassium chlorite or sodium chlorite; or a persulfuric acid compound, potassium persulfate or sodium persulfate; or an organic oxidizing agent such as a reagent used in DMSO oxidation (a complex of dimethyl sulfoxide with dicyclohexylcarbodiimide, oxalyl chloride, acetic anhydride or phosphorous pentaoxide, or a complex of pyridine-sulfur trioxide); a peroxide such as tbutylhydroperoxide; a stable cation such as triphenylmethyl cation; a succinic acid imide such as N-bromosuccinic acid P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 108 imide; a hypochlorous acid compound such as t-butyl hypochlorite; an azodicarboxylic acid compound such as methyl azodicarboxylate; a disulfide such as dimethyl disulfide, diphenyl disulfide or dipyridyl disulfide and triphenylphosphine; a nitrous acid ester such as methyl nitrite; 00 a carbon tetrahalide, carbon tetrabromide; or a quinone V' compound, 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ); 00 preferably iodine molecule.
c The deoxidizer to be used can be a heterocyclic amine O such as pyridine or dimethylaminopyridine; or an aliphatic amine (C such as trimethylamine, triethylamine or diisopropylethylamine; and is preferably an aliphatic amine (particularly diisopropylethylamine). The reaction temperature is not particularly limited but it is normally from -50 to 50 0
C,
preferably room temperature.
The reaction time varies depending on the starting materials, the reagent and the temperature used, but it is normally from 5 minutes to 30 hours, preferably in the case where the reaction is carried out at room temperature, it is minutes.
The solvent in the reaction for forming a methoxyethylamino phosphate group is not particularly limited so long as it does not inhibit the reaction, but carbon tetrachloride that is normally used as a reagent is used at a solvent amount.
The reaction temperature is not particularly limited in a range of from -50 to 100 0 C, but in the case where the reaction is carried out at room temperature, the reaction time is from 1 to 10 hours.
Further, in the case where the phosphodiester compound or (10) obtained in Step A-3 or B-4 is condensed to form the phosphate tri-ester bond in the present step, the solvent used in the present step is not particularly limited so long as it does not inhibit the reaction, but an aromatic amine such as pyridine is preferably used.
The condensing agent used in the condensation can be P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 dicyclocarbodiimide (DCC), mesitylenesulfonic chloride (Ms-Cl), triisopropylbenzenesulfonic chloride, mesitylenesulfonic acid triazolide (MST), mesitylenesulfonic acid-3-nitrotriazolide (MSNT), triisopropylbenzenesulfonic acid tetrazolide (TPS-Te), triisopropylbenzenesulfonic acid nitroimidazolide (TPS-NI) or triisopropylbenzenesulfonic acid pyridyltetrazolide, and is preferably MSNT, TPS-Te and TPS-NI.
The reaction temperature is not particularly limited in a range of from -10 to 100 0 C, but the reaction is normally carried out at room temperature.
The reaction time varies depending on the solvent used and the reaction temperature, but in the case where pyridine is used as the reaction solvent, and the reaction is carried out at room temperature, it is 30 minutes.
Cleavage from CPG in the case where the 2-5 analog is bonded to CPG and removal of the protecting groups other than the substituent portion at the 5'-end described next can be carried out by a publicly known method Am. Chem. Soc., 103, 3185, (1981)).
The resulting crude 2-5A analog can be confirmed by purification using a reverse phase chromatocolumn and analyzing the purity of the purified product by HPLC.
The chain length of the thus obtained oligonucleotide analog is normally from 2 to 50, preferably from 10 to nucleoside units.
(Step G-l) The present step is to prepare 2-5A analog on a DNA automatic synthesizer by ordinary methods using CPG using the compounds (12) or (14) prepared in Step A-i, A-2, A-3, B-2, B-3, B-4, C-1 or D-l and (21).
CPG (22) is the same as the compound (24) described in Process G of Japanese Patent Application (Kokai) No. 2002-249497, and the present step is carried out similarly to Step F-1.
S :/ChemicaUSankyo/FP200343/FP200343s L~doc P89346/FP-200343/gds-mglspecification//03.05.05 (Step H-1) The present step is a step, wherein 2-5A analog is produced on a DNA automatic synthesizer by ordinary methods using CPG using the compounds (12) or (14) prepared in Step A-i, A-2, A-3, B-2, B-3, B-4, C-1 or D-1 and (21).
CPG (23) is the same as the compound described in Japanese Patent Application (Kokai) No. Hei 7-53587, and the present step is carried out similarly to Step F-1.
Further, in the 2-5A analog in the case where any one of R 1
R
2 R R R R 6 and R 7 is a mercapto group, after the 2-5A analog is synthesized and purified by Process F, G or H, a substituent can be introduced onto the mercapto group by reacting with a compound having a halide group, in the presence of a base in an inert solvent.
The halogen can be, for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a chlorine atom, a bromine atom, or an iodine atom.
The compound having a halide group to be used is not particularly limited so long as it is a compound having a halide group which can be reacted with a thiophosphoric acid group, and can be, for example, an "alkyl halide which may be substituted" such as an ethyl halide, a propyl halide, a butyl halide, a 2halo ethanol, a 3-halo propanol, or a 4-halo butanol; an "acyloxyalkyl halide" such as a 2-(stearoyloxy)ethyl halide, a 2-(myristoyloxy)ethyl halide, a 2-(decanoyloxy)ethyl halide, a 2-(benzoyloxy)ethyl halide, a 2-(pivaloyloxy)ethyl halide, a 2- (2,2-dimethyloctadecanoyloxy)ethyl halide, a 3- (stearoyloxy)propyl halide, a 3-(myristoyloxy)propyl halide, a 3-(decanoyloxy)propyl halide, a 3-(benzoyloxy)propyl halide, a 3-(pivaloyloxy)propyl halide, a 3-(2,2dimethyloctadecanoyloxy)propyl halide, a 4-(stearoyloxy)butyl halide, a 4-(myristoyloxy)butyl halide, a 4-(decanoyloxy)butyl halide, a 4-(benzoyloxy)butyl halide, a 4-(pivaloyloxy)butyl halide, or a 4-(2,2-dimethyloctadecanoyloxy)butyl halide; an "alkylcarbamoyloxyalkyl halide" such as a 2- S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 111 stearylcarbamoyloxyethyl halide; or one of the following compounds: 0 0
H
0
H
0
H
s 0
H
0
HH
H H In the above compounds, a 2-stearoyloxyethyl halide and a 2- (2,2-dimethyloctadecanoyloxy)ethyl halide are preferred.
Of the compounds having these halide groups, compounds having an ester group or a carbamate group or an amide group or a thio ester group or a urea group a thiocarboxylic acid ester group or or a thiocarboxylic acid amide group or can be prepared in the presence of a base or a condensing agent by condensation of an acid halide compound or a carboxylic acid compound with a compound having an alcohol group; condensation of a formic acid ester halide compound with a compound having an amino group; condensation of an acid halide compound or a carboxylic acid compound with a compound having an amino group; condensation of an acid halide compound or a carboxylic acid compound with a compound having a thiol group; condensation of compounds having two kinds of amino group with phosgene; condensation of a thiocarboxylic acid compound with a compound having an alcohol group; or condensation of a thiocarboxylic acid compound with the compound having an amino .group.
The base to be used can be a heterocyclic amine such as pyridine or dimethylaminopyridine; or an aliphatic amine such as S :/ChemicaUSankyo/FP200343/FP200343s L~doc P89346/FP-200343gds-mg/specification//103.05.5 trimethylamine, triethylamine or diisopropylamine; and is preferably a heterocyclic amine (particularly pyridine).
There is no particular limitation on the solvent to be used, provided that it does not inhibit the reaction and dissolves the starting material to a certain extent, and it can be water; an amide such as dimethylformamide, dimethylacetamide or hexamethylphosphortriamide; a sulfoxide such as dimethyl sulfoxide; a heterocyclic amine such as pyridine; a nitrile such as acetonitrile; or a mixture of these solvents; and is preferably dimethylformamide.
The reaction temperature is not particularly limited in a range of from -50 to 100 0 C, but the reaction is normally carried out at room temperature. The reaction time varies depending on the material, the reagent used, and the temperature, but it is normally from 10 hours to 100 hours.
The reaction speed can also be appropriately increased by adding an iodide salt such as tetrabutylammonium iodide.
Instead of using CPG(23) used in method H, a antisense oligonucleotide can be synthesized by condensing a phosphoramidite serving as a linker, such as DMT-butanol-CED phosphoramidite (ChemGene) or Spacer phosphoramidite 18 (GlenResearch), to CPG to which is bonded an oligonucleotide having the desired antisense sequence that is protected with a protecting group, followed by carrying out the procedure of the present step. For example, in the case of "CPG to which is bonded an oligonucleotide protected with a protecting group", a modified oligonucleotide can be synthesized in which the oxygen atom at the 2' position of the sugar portion is bridged to a carbon atom at the 4' position with an alkylene group according to the method described in Japanese Patent Application (Kokai) No. Hei 10-304889 or Japanese Patent Application (Kokai) No.
2000-297097. In addition, a modified oligonucleotide having a 2'-O-methoxyethoxy group can be synthesized by referring to the literature (Teplove, M. et al., Nat. Struct. Biol. (1999), 6, 535; Zhang H. et al., Nature Biotech. (2000), 18, 862), and a modified oligonucleotide having a 3'-amino group can be S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 synthesized by referring to the literature (Gryaznov, S.M. et al., Proc. Natl. Acad. Sci. USA 1995, 92, 5798; Tereshko, V. et al., J. Am. Chem. Soc. 1998, 120, 269).
The antitumor activity (cytocidal activity) of the present compounds can be investigated by adding the present compounds to cancer cells in a medium, and culturing the cells, followed by counting the number of viable cells using the MTT assay method (Tim Mosmann, J. Immunological Methods, 1983: 55-63), the MTS assay method (Rotter, Thompson, B.K., Clarkin, Owen, T.C. Nat. Toxins 1993; 303-7), the XTT assay method (Meshulam, Levitz, Christin, Diamond, R.D. J. Infect. Dis. 1995; 172(4): 1153-6), or Trypan blue staining.
The antivirus activity of the present compounds can be investigated using an infected cell culture system such as HeLa cells, MDCK cells, MRC-5 cells or the like, by adding the present compounds to virus cells, such as of vaccinia virus, influenza virus or cytomegalovirus, in a medium either before or after infection, culturing for a predetermined amount of time, and then measuring the virus growth inhibition rate using the plaque assay method which measures virus infection titer (Kobayashi, Nagata, K. Virus Experimental Protocols, Medical View Publishing), or the ELISA method which measures the level of virus antigen (Okuno, Tanaka, Baba, Maeda, A., Kunita, Ueda, J. Clin. Microbiol., June 1, 1990; 28(6): 1308-13).
The .administration forms of the 2-5A analogs of general formula of the present invention can include, for example, oral administration by tablets, capsules, granules, powders or syrups, or parenteral administration by injection or suppositories. These preparations are prepared by known methods using additives such as excipients (which include, for example, organic excipients such as sugar derivatives, lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives, corn starch, potato starch, a-starch and dextrin; cellulose derivatives, crystalline cellulose; gum arabic; S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 114 dextran; and pullulan; and inorganic excipients such as silicate derivatives, light silicic anhydride, synthetic aluminum silicate, calcium silicate and magnesium aluminate metasilicate; phosphates, calcium hydrogenphosphate; carbonates, calcium carbonate; and sulfates, calcium 00 sulfate), lubricants (which can include, for example, stearic l acid and its metal salts such as stearic acid, calcium stearate 00 and magnesium stearate; talc; colloidal silica; waxes such as Sbeeswax and spermaceti; boric acid; adipic acid; sulfates such O as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL- (CN leucine; sodium salts of aliphatic acids; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride; and the above starch derivatives), binders (which can include, for example, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, Macrogol, and compounds similar to the above excipients), disintegrating agents (which can include, for example, cellulose derivatives such as low substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium and internally cross-linked carboxymethyl cellulose sodium; and chemically modified starches/celluloses such as carboxymethyl starch, carboxymethyl starch sodium and cross-linked polyvinylpyrrolidone), stabilizers (which can include paraoxybenzoates such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (which can include, for example, sweeteners, acidifiers, perfumes or the like normally used), diluents, and the like.
While the amount used varies depending on the symptoms, the age, the administration method, and the like, it is desirable to administer once to several times a day, and, in the case of oral administration, 0.01 mg/kg body weight (preferably 0.1 mg/kg body weight) per time as a lower limit and 1000 mg/kg body weight (preferably 100 mg/kg body weight) as an upper limit, P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 and, in the case of intravenous administration, 0.001 mg/kg body weight (preferably 0.01 mg/kg body weight) per time as a lower limit and 100 mg/kg body weight (preferably 10 mg/kg body weight) as an upper limit corresponding to the symptoms.
Further, the preparations may be used in combination with other antitumor agents, for example, nitrosourea type chemicals such as 5FU, AraC, ACNU or BCNU, cisplatin, daunomycin, adriamycin, mitomycin C, vincristine, and taxol.
In the following, the present invention will be explained in more detail by Examples, Reference examples and Test examples.
Best Mode for Carrying out the Invention (Example 1) Synthesis of Example 1 compound (Exemplary Compound No. 4)
NH
NH2 HO 0 O- ON NH2 SNH2 OHO S=P-OH N Example 1 compound OH OH The ABI Model 392 DNA/RNA Synthesizer (Applied Biosystems) was used as the DNA synthesizer. The solvents, reagents, and phosphoramidite concentrations in each synthesis cycle were the same as in the case of general natural oligonucleotide synthesis, and the products of Applied Biosystems were used for those reagents and solvents other than the phosphoramidite and sulfurizing agent. The riboadenosine analog, Bz-Adenosine-RNA-500 (Glen Research) pmol), bound to a CPG support, was used as the starting substance. Synthesis was carried out using the DNA synthesizer S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 2, while the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 3. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1 and 2, while iodine was used in cycle 3.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Sulfurization (cycles 1 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
Oxidation (cycle Iodine/water/pyridine/tetrahydrofuran; sec.
After synthesizing the protected 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 0-13% CH 3 CN (linear gradient, 30 min.); 40 0 C; 10 ml/min; 254 nm), and the fractions that eluted at 20.9, 22.7, 25.4 and 28.0 S :/Chemical/Sankyo/FP200343/FP200343s L~doc P89346/FP-200343/gds-mg/specification//03.05.O minutes corresponding to the four diastereomers were collected.
The present compound eluted in the vicinity of 10.55 minutes when analyzed by ion exchange HPLC (column (Tosoh DEAE-2SW (4.6 x 150 solution A (20% acetonitrile), solution B acetonitrile and 67 mM phosphate buffer, 2 M NaC1); solution B 5->60% (15 min., linear gradient); 60 0 C; 1 ml/min). (Yield: 457 nmol as UV measured value using the calculated e 39400 (260 nm) of the adenosine trimer)) kmax (H 2 0) 258.3 nm, ESI-Mass (negative): 1080.1 (Example 2) Synthesis of Example 2 compound (Exemplary Compound No. 1)
NH
2 N
N
II
HO-P-O-i OH 0
NH
2 OMeO N O=P-OH N N N NH 2 OH0 <0 l O=P-OH NJN Example 2 compound O Synthesis was carried out using the compound of Example 17 described in Japanese Patent Application (Kokai) No. 2002- 249497 (2.0 pmol) as the 5'-O-DMTr-riboadenosine analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycle 1, 5'-DMT-3'-(O-methyl) adenosine(bz)2'-phosphoramidite (ChemGene) was used in cycle 2, and Chemical Phosphorylation Reagent II (Glen Research) was used in cycle 3. For the oxidation or sulfurizing agent, iodine was S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 used in cycles 1 and 2, while xanthane hydride (Tokyo Kasei Kogyo) was used in cycle 3.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 1 and 2): lodine/water/pyridine/tetrahydrofuran; 15 sec.
Sulfurization (cycle Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS x 250 0.1 M aqueous triethyl amine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, 30 min.); 40 0 C; 10 ml/min; 254 nm), and the fraction that eluted at 16.7 minutes was collected.
The present compound eluted in the vicinity of 9.46 minutes when analyzed by reverse HPLC (column (Tosoh superODS (4.6 x 50 mm)); 0.1M aqueous triethylamine acetate (TEAA), pH7; 0-25% CH 3
CN
(linear gradient, 14 min); 60 0 C; 10 ml/min). (Yield: 445 nmol as UV measured value at 260 nm) kmax (H20) 258.2 nm, ESI-Mass (negative): 1074.15 S :/Chemical/Sankyo/FP00343/FP00343s L~doc P89346/FP-200343/gds-mgfspcification//03.05.O S (Example 3) Synthesis of Example 3 compound (Exemplary Compound No.
NHH
<1 HON:] N NH2 OH O NH2 S=P-OH N OH 0 S=P-OH Example 3 compound OH OH Synthesis was carried out using Bz-Adenosine-RNA 500 (Glen Research Co.) (2.0 pmol) as the analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 2, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 3.
Xanthane hydride (Tokyo Kasei Kogyo) was used as the sulfurizing agent in cycles 1, 2 and 3.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 1, 2 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure.with the 5'-DMTr group still intact, together with cleaving the oligomer from the support, the cyanoethyl group S:/ChemicaYSankyo/FP200343/FP200343s1 .doc P89346/FP-200343/gds-mg/specification//03.05.05 120 serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol i The solvent was then distilled off under reduced pressure and the product was purified by reverse phase HPLC (Shimadzu 00 Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 x 250 It) 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 00 CH 3 CN (isocratic); 40 0 C; 10 ml/min; 254 nm), and the fractions
(N
C that eluted at 9.5 and 11.8 minutes as diastereomers were 0 collected. After the solvent was distilled off under reduced pressure, 80% aqueous acetic acid was added thereto, the mixture was left to stand for 30 minutes, and the DMTr group was removed. After the solvent was distilled off, a mixture of concentrated aqueous ammonia-ethanol was added thereto, and the mixture was left to stand for 30 minutes. After the solvent was distilled off, the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 followed by reacting for hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, 30 min.); 40 0 C; 10 ml/min; 254 nm), and the fractions that eluted at 16.5-19.1 minutes were collected. The present compound eluted in the vicinity of 8.8-9.8 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 mm)); 0.1M aqueous triethylamine acetate (TEAA), pH7; 0-25% CH 3
CN
(linear gradient, 14 min); 60 0 C; 1 ml/min). (Yield: 565 nmol as UV measured value at 260 nm) kmax (H 2 0) 258.2 nm, ESI-Mass (negative): 1096.1 (Example 4) Synthesis of Example 4 compound (Exemplary Compound No. 8) P:\OPER\PDB\Speci\200324578 Ispadoc 7/7/05 121
NH
S N N
HO-P-O-I
OH NH 2 1T N OH
<N
S=P-OH NN
NH
2 OH
N
S=P-OH N Example 4 compound OH OH Synthesis was carried out using Bz-Adenosine-RNA 500 (Glen Research Co.) (2.0 pnol) as the analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 2 and Chemical Phosphorylation Reagent II (Glen Research Co.) was used in cycle 3. Xanthane hydride (Tokyo Kasei Kogyo) was used as the sulfurizing agent in cycles 1, 2 and 3.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 1, 2 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous S:/Chemical/Sankyo/FP200343/FP200343sl .doc P89346/FP-200343/gds-mg/specification//03.O.05 ammonia and ethanol The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 6-25% CH 3 CN (linear gradient, 30 min.); 40 0 C; 10 ml/min; 254 nm), and the four fractions that eluted at 13.3, 13.7, 13.9 and 14.4 minutes corresponding to the four diastereomers were collected. The present compound eluted in the vicinity of 7.2minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-20% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min). (Yield: 252 nmol as UV measured value at 260 nm) Xmax
(H
2 0) 258.0 nm, ESI-Mass (negative): 1052.1 (Example Synthesis of Example 5 compound (Exemplary Compound No. 290)
NH
OH H OJ N O =P-OH N Exanple 5 compoud (Exempary Compound No. 290) nmol of Example 2 compound were dissolved in 30 [l of anhydrous DMF, and 1 p1 of pivaloyloxymethyl chloride (Tokyo Kasei Kogyo), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 p1 of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 p1 of water were added, and the aqueous layer was washed three times with S:/ChemicaISankyoIFP200343/FP200343s I.doc P89346/FP-200343/gds-mg/specification//03050 123 il of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-42% CH3CN (linear gradient, min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 5.6 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 mm)); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-42% CH 3
CN
(linear gradient, 14 min); 60 0 C; 1 ml/min), it eluted at 7.52 minutes. Yield: 4.8 nmol, kmax (H20) 258 nm, ESI-Mass (negative); 1188.2 (Example 6) Synthesis of Example 6 compound (Exemplary Compound No. 334) NH2 0 O H NH, 0=P-OH N1N) OH 0 N 0=P-OH NN Examplo 6 compound (Exemplary Compound No. 334) nmol of Example 2 compound were dissolved in 30 il of anhydrous DMF, and 1 p4 of thioacetic acid S-(2-bromo-ethyl) ester (Bauer, L. et al. J. Org. Chem. 1965, 30, 949-951), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 il of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 pi of water were added, and the aqueous layer was washed three times with 50 il of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), S:/Chemical/Sankyo/FP200343/FP200343s 1 .doc P89346/FP-200343/gds-mg/specification//03.05.05 and the fraction at 4.5 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 7.25 minutes. Yield: 14 nmol, kmax (H 2 0) 258 nm, ESI-Mass (negative); 1176.2 (Example 7) Synthesis of Example 7 compound (Exemplary compound No. 953) SH N N =P-OP N
N
OH J
O=P-OH
Example 7 compond (Exemplary Conpound No. 953) Synthesis was carried out using the compound of Example 17 described in Japanese Patent Application (Kokai) No. 2002- 249497 (2.0 pmol) as the 5'-O-DMTr-riboadenosine analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycle 1, 5'-DMT-3'-(O-methyl) adenosine(N-bz)2'-phosphoramidite (ChemGene) was used in cycle 2, and Chemical Phosphorylation Reagent II (Glen Research) was used in cycle 3. For the oxidation or sulfurizing agent, iodine was used in cycle 1, and xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 2 and 3.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic S :/ChemicaSankyo/FP200343/FP2O343s I.doc P89346/FP-200343/gds-mg/specification//03.05.05 anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycle lodine/water/pyridine/tetrahydrofuran; sec.
sulfurization (cycles 2 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 20 min.); 40 0 C; 10 ml/min; 254 nm), and the fractions that eluted at 12.1 and 13.0 minutes corresponding to the two diastereomers were collected. The present compound eluted in the vicinity of 8.66 and 8.98 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqeous triethylamine acetate (TEAA), pH 7; 0-15%
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min). Yield: 768 nmol as UV measured value at 260 nm, kmax (H 2 0) 258 nm, ESI- Mass (negative): 1090.2 (Example 8) Synthesis of Example 8 compound (Exemplary Compound No. 954) S:/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mg/specification//03.05.05 Eample 8 compound (Exemplay Compound No. 954) Synthesis was carried out using the compound of Example 17 described in Japanese Patent Application (Kokai) No. 2002- 249497 (2.0 Wmol) as the 5'-O-DMTr-riboadenosine analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 munol of RNA. 3'tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 2, and Chemical Phosphorylation Reagent II (Glen Research) was used in cycle 3.
For the oxidation or sulfurizing agent, iodine was used in cycle 1, and xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 2 and 3.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycle lodine/water/pyridine/tetrahydrofuran; sec.
sulfurization (cycles 2 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 ammonia and ethanol The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 20 min.); 40 0 C; 10 ml/min; 254 nm), and the fractions that eluted at 11.5 and 12.4 minutes corresponding to the two diastereomers were collected. The present compound eluted in the vicinity of 8.28 and 8.60 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min). Yield: 718 nmol as UV measured value at 260 nm, Xmax (H 2 0) 258 nm, ESI- Mass (negative): 1076.1 (Example 9) Synthesis of Example 9 compound (Exemplary Compound No. 955)
NH
2 0 H
NH,
Example 9 conound (Eemplary Compound No. 955) nmol of Example 7 compound were dissolved in 30 il of anhydrous DMF, and 1 il of pivaloyloxymethyl chloride (Tokyo Kasei Kogyo), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 il of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 gl of water were added, and the aqueous layer was washed three times with S:/Chemical/Sankyo/FP200343/FP200343s I.doc P89346/FP-200343/gds-mg/specification//03.05.05 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, min); 60 0 C; 2 ml/min; 254 nm), and the fractions at minutes and 5.6 minutes corresponding to two diastereomers were collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, min); 60 0 C; 1 ml/min), it eluted at 7.42 and 7.56 minutes.
Yield: 1.8 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258 nm, ESI-Mass (negative); 1204.2 (Example Synthesis of Example 10 compound (Exemplary Compound No. 956)
N
o s- o o.Aj 'N N.z
OH
Eaple 10 compound (Exemplary Compound No. 956) nmol of Example 8 compound were dissolved in 30 p1 of anhydrous DMF, and 1 1l of pivaloyloxymethyl chloride (Tokyo Kasei Kogyo), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo),. and 1 pl of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 .1 of water were added, and the aqueous layer was washed three times with pl of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, S:/ChemicaUSankyo/FP200343/FP00343sI .doc P89346/FP-200343/gds-mg/specification/03.05.05 min); 60 0 C; 2 ml/min; 254 nm), and the fractions at 5.7 and 5.9 minutes corresponding to two diastereomers were collected.
When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, min); 60 0 C; 1 ml/min), it eluted at 7.68 and 7.85 minutes.
Yield: 11 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258 nm, ESI-Mass (negative); 1191.20 (Example 11) Synthesis of Example 11 compound (Exemplary Compound No. 957)
NH,
S N N 0 OH
NH
O
0=-SH N N
NH
2 OH N J 0SP-OH Example 11 compound (Exmplary Compoud No. 957) nmol of Example 7 compound were dissolved in 30 gl of anhydrous DMF, and 1 gl of thioacetic acid S-(2-bromo-ethyl) ester (Bauer, L. et al. J. Org. Chem. 1965, 30, 949-951), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 p. of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 1l of water were added, and the aqueous layer was washed three times with 50 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fractions at 4.7 and 4.8 minutes corresponding to two diastereomers were collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x S:/Chemical/Sankyo/FP200343/FP200343s 1 .doc P89346/FP-200343/gds-mg/specification//03.05.05 130 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43%
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 6.57 and 6.75 minutes. Yield: 20 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 258 nm, ESI-Mass (negative); 1192.1 (Example 12) Synthesis of Example 12 compound (Exemplary Compound No. 958)
NH,
O N 9 1 o 1 0 H NH, OH P oj ON 0 O=P-OH -N Eample 12 compound (Eempary Compound No. 958) nmol of Example 8 compound were dissolved in 30 il of anhydrous DMF, and 1 p1 of thioacetic acid S-(2-bromo-ethyl) ester (Bauer, L. et al. J. Org. Chem. 1965, 30, 949-951), approximately 1 mg of tetrabutylammonium iodide (Tokyo Kasei Kogyo), and 1 p. of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 gl of water were added, and the aqueous layer was washed three times with 50 p. of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fractions at 4.9 and 5.1 minutes corresponding to two diastereomers were collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-43%
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 6.83 and 7.04 minutes. Yield: 6.7 nmol in terms of UV S :/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mg/specification//03.05.05 measurement at 260 nm, X max (H 2 0) 258 nm, ESI-Mass (negative); 1178.1 (Example 13) Synthesis of Example 13 compound (Exemplary Compound No. 964)
N
0 OH N NH N
NO
O=P-OH N ON
N
O=P-OH
Exampe 13 compound (Exemply Compound No. 964) nmol of Example 2 compound were dissolved in 30 il of anhydrous DMF, and 1 pl of 2-(pivaloyloxy)ethyl bromide (Preparation process described in EP0395313), and 1 pl of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 p1 of water were added, and the aqueous layer was washed three times with 50 pl of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 60 0 C; 5-80% CH 3
CN
(linear gradient, 10 min); 600C; 2 ml/min; 254 nm), and the fraction at 4.3 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 7.48 minutes. Yield: 19.1 nmol in terms of UV measurement at 260 nm, X max (H 2 0) 258.6 nm, FAB-Mass (negative); 1202 (Example 14) Synthesis of Example 14 compound (Exemplary Compound No. 965) S:/Chemical/Sankyo/FP200343/FP200343sl.doc P89346/FP-200343/gds-mg/specification//03.05.05 132 Example 14 copound (Exempary Compound No. 965) nmol of Example 2 compound were dissolved in 30 pl of anhydrous DMF, and 1 il of 2-(benzoyloxy)ethyl bromide (Tokyo Kasei Kogyo), and 1 |l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight.
After completion of the reaction, 100 pg of water were added, and the aqueous layer was washed three times with 50 il of AcOEt.
After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 7.0 minutes was collected.
When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80% CH 3 CN (linear gradient, min); 60 0 C; 1 ml/min), it eluted at 7.44 minutes. Yield: 19.7 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 258.7 nm, FAB-Mass (negative); 1222 (Example Synthesis of Example 15 compound (Exemplary Compound No. 967) S:/Chemical/Sankyo/FP200343/FP200343s L~doc P89346/FP-200343/gds-mg/specification//03.05.05 Example 15 Compound (Exemplary Compound No. 967) nmol of Example 2 compound were dissolved in 30 1l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 il of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 p1 of water were added, and the aqueous layer was washed three times with 50 1l of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 8.2 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80%
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 14.62 minutes. Yield: 14.9 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 260.1 nm, FAB-Mass (negative); 1384 (Example 16) Synthesis of Example 16 compound (Exemplary Compound No. 968) S:/ChemicaSankyo/FP200343/FP200343s L~doc P89346/FP-200343/gds-mg/specification//03.O.05 Example 16 compound (Exemplary Compound No. 968) nmol of Example 2 compound were dissolved in 30 p1 of anhydrous DMF, and 1 mg of 2-(myristoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 pi of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 p of water were added, and the aqueous layer was washed three times with 50 pl of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 6.3 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80%
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 12.57 minutes. Yield: 13.1 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 259.7 nm, FAB-Mass (negative); 1328 [M-HI-.
(Example 17) Synthesis of Example 17 compound (Exemplary Compound No. 969) S :/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mg/specification/03.05.05 Example 17 compound (Exemplary Compomd No. 969) nmol of Example 2 compound were dissolved in 30 p1 of anhydrous DMF, and 1 mg of.2-(decanoyloxy)ethyl bromide (Devinsky, Ferdinand et al., Collect. Czech. Chem. Commun. 49, 12, 1984, 2819-2827), and 1 .1 of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 l1 of water were added, and the aqueous layer was washed three times with 1l of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80% CH 3 CN (linear gradient, min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 4.3 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 mm)); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-80% CH 3
CN
(linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 10.36 minutes. Yield: 19.8 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258.2 nm, FAB-Mass (negative); 1272 (Example 18) Synthesis of Example 18 compound (Exemplary Compound No. 1074) S :/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mg/specification/03.05.05 136 00
S-OH
00 Example 18 compound S(Exemplary Compound No. 1074) 0 Synthesis was carried out with the DNA synthesizer based pC on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-Phosphate CPG (Glen Research) (2.0 pmol) was used as the solid phase carrier. The compound of Example 16 described in Japanese Patent Application (Kokai) No. 2002-249497 was used as the phosphoramidite in cycle 1, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycle 2, 5'-DMT-3'-(O-methyl) adenosine(Nbz)2'-phosphoramidite (ChemGene) was used in cycle 3, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycle 1, and iodine was used in cycles 2, 3 and 4.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 2, 3 and 4): lodine/water/pyridine/tetrahydrofuran; 15 sec.
sulfurization (cycle Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 300C to remove the DMTr group and the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 5-17% CH 3 CN (linear gradient, min.); 40 0 C; 10 ml/min; 254 nm), and the fraction that eluted at 14.9 minutes was collected. The present compound eluted in the vicinity of 6.77 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, min); 600C; 1 ml/min). Yield: 1440 nmol as UV measured value at 260 nm, Xmax (H 2 0) 258.5 nm, ESI-Mass (negative): 1198.1 (Example 19) Synthesis of Example 19 compound (Exemplary Compound No. 1075) NH
N
0 NY
OH
Examle 19 compound (Exmplary Compound No. 1075) Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol S :/ChemicaUSanyoFP00343/FP200343s L~doc P89346/FP-200343/gds-mg/specification//03.O5.05 of RNA. 3'-Phosphate CPG (Glen Research) (2.0 pmol) was used as the solid phase carrier. The compound of Example 16 described in Japanese Patent Application (Kokai) No. 2002-249497 was used as the phosphoramidite in cycle 1, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycle 2, 5'-DMT-3'-(O-methyl) adenosine(Nbz)2'-phosphoramidite (ChemGene) was used in cycle 3, and Chemical Phosphorylation Reagent II (Glen Research) was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1 and 4, and iodine was used in cycles 2 and 3.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 2 and 3): lodine/water/pyridine/tetrahydrofuran; 15 sec.
sulfurization (cycles 1 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 30 0 C to remove the DMTr group and the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS (20 x 250 0.1 M aqueous S :/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mglspecification//03.05.05 triethylamine acetate (TEAA), pH 7; 5-17% CH 3 CN (linear gradient, min.); 40 0 C; 10 ml/min; 254 nm), and the fraction that eluted at 15.5 minutes was collected. The present compound eluted in the vicinity of 8.63 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, min); 60 0 C; 1 ml/min). Yield: 1482 nmol as UV measured value at 260 nm, Xmax (H 2 0) 258.2 nm, ESI-Mass (negative): 1170.1 (Example Synthesis of Example 20 compound (Exemplary Compound No. 1937) 0=P-OH
SH
Exsamoplo 20 conpound (Exemplary Compound No. 1937) Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-Phosphate CPG (Glen Research) (1.0 pmol) was used as the solid phase carrier. 5'-DMT-3'-(O-methyl) adenosine(Nbz)2'-phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 3, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycle 2, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4.
Iodine was used as the oxidizing agent.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
S:/ChemicalSankyo/FP200343/FP200343s 1 .doc P89346/FP-200343/gds-mg/specification//03.05.05 3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation: Iodine/water/pyridine/tetrahydrofuran; 15 sec.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 30 0 C to remove the DMTr group and the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 2.5-10% CH 3 CN (linear gradient, 10 min.); 60 0 C; 2 ml/min), and the fraction that eluted at 4.8 minutes was collected. The present compound eluted in the vicinity of 3.16 minutes when analyzed by reverse phase HPLC (column (((Merck chromolith (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-10% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min). Yield: 95 nmol as UV measured value at 260 nm, Xmax
(H
2 0) 256.2 nm, ESI-Mass (negative): 1171.9 (Example 21) Synthesis of Example 21 compound (Exemplary Compound No. 1099) S:/Chemical/SankyoIFP200343/FP200343s I.doc P89346/FP-200343/gds-mg/specification//03050 Exampe 21 compound (Exemplary Compound No. 1099) Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-Phosphate CPG (Glen Research) (1.0 pmol) was used as the solid phase carrier. 5'-DMT-3'-(O-methyl) adenosine(Nbz)2'-phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 3, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycle 2, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4.
For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1 and 2, and iodine was used in cycles 3 and 4.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 3 and 4): lodine/water/pyridine/tetrahydrofuran; 15 sec.
sulfurization (cycles 1 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification/03.05.05 phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure, and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue, followed by reacting for 5 hours at 30 0 C to remove the DMTr group and the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 0.1 M aqueous triethyl amine acetate (TEAA), pH 7; 5-10% CH 3 CN (linear gradient, 10 min.); 60 0 C; 2 ml/min), and the fractions that eluted at 6.0 and 6.4 minutes were collected. The present compound eluted in the vicinity of 4.89 and 5.43 minutes when analyzed by reverse phase HPLC (column (((Merck chromolith (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-10% CH 3 CN (linear gradient, min); 60 0 C; 2 ml/min). Yield: 54 nmol as UV measured value at 260 nm, kmax (H 2 0) 258.0 nm, ESI-Mass (negative): 1189 (Example 22) Synthesis of Example 22 compound (Exemplary Compound No. 1110) H NH f
N
O=OH 9 0= OH Example 22 compound (Exemplary Compound No. 1110) 500 mg (1.6 mmol) of 2,2-Dimethyl-octadecanoic acid (Roth, Bruce D. et al., J. Med. Chem. 1992, 35(9), 1609-17) were dissolved in anhydrous dichloromethane (10 ml), and 350 mg (1.8 mmol) of dicyclohexylcarbodiimide (DCC), and 140 il (2 mmol) of 2-bromoethanol were added thereto, followed by stirring of the S:/Chemical/Sankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 mixture at room temperature overnight. The reaction mixture was purified using a silica gel column (elution by hexane-ethyl acetate solvent mixture) to obtain 230 mg of 2-(2,2dimethyloctadecanoyloxy)ethyl bromide to be used below.
100 nmol of Example 19 compound were dissolved in 100 il of anhydrous DMF, and 3 mg of 2-(2,2dimethyloctadecanoyloxy)ethyl bromide, and 3 1l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 pl of water were added, and the aqueous layer was washed three times with 200 1l of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 33-80% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 6.7 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH3CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 9.65 minutes. Yield: 24.5 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 259.3 nm, ESI-Mass (negative); 1537.3 (Example 23) Synthesis of Example 23 compound (Exemplary Compound No. 1111)
NH,
oH1SI
NH
0,H9, C< N
I
O=P-OH N 0=P-OH N Example 23 compound (Exemplary Compound No. 1111) 100 nmol of Example 19 compound were dissolved in 100 p1 of anhydrous DMF, and 3 mg of 2-(stearoyloxy)ethyl bromide S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 p of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 p1 of water were added, and the aqueous layer was washed three times with 200 p. of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 52-100%
CH
3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 1.9 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 15-100% CH 3 CN (linear gradient, 10 min); 600C; 1 ml/min), it eluted at 10.06 minutes. Yield: 39.8 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258.2 nm, ESI-Mass (negative); 1508.4 (Example 24) Synthesis of Example 24 compound (Exemplary compound No. 1112)
H
OH 1 NH, O=P-O H (Exmphlry Coound No. 1112) 100 nmol of Example 19 compound were dissolved in 100 p.
of anhydrous DMF, and 3 mg of 2-(myristoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 pg of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 pi of water were added, and the aqueous layer was washed three times with 200 pi of AcOEt. After the aqueous S:/Chemical/Sankyo/FP200343/FP200343s 1.doc P89346/FP-200343/gds-mg/specification//03.05.05 layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 33-100%
CH
3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 4.1 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 15-100% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 8.75 minutes. Yield: 54.3 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 258.0 nm, ESI-Mass (negative); 1452.4 (Example Synthesis of Example 25 compound (Exemplary Compound No. 1113)
NH,
OH NH2 (Exmply Compond No. 1113) 100 nmol of Example 19 compound were dissolved in 100 pl of anhydrous DMF,-and 3 mg of 2-(decanoyloxy)ethyl bromide (Devinsky, Ferdinand et al., Collect. Czech. Chem. Commun. 49, 12, 1984, 2819-2827), and 3 Il of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 il of water were added, and the aqueous layer was washed three times with 200 gl of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 15-62% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 6.2 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 0.1M aqueous triethylamine acetate (TEAA), pH 7; 15-100%
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 7.29 minutes. Yield: 50.9 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258.4 nm, ESI-Mass (negative); 1396.3 (Example 26) Synthesis of Example 26 compound (Exemplary Compound No. 1938)
NH,
OMe9 OHM9
P-OH
Examle 26 compound (Exemplay Compound No. 1938) nmol of Example 20 compound were dissolved in 30 .1 of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 p1 of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 Jl of water were added, and the aqueous layer was washed three times with 100 1l of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 28-100%
CH
3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 5.3 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column Merck chromolith (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 7.51 minutes. Yield: 14.9 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 258 nm, ESI-Mass S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 (negative); 1483.2 (Example 27) Synthesis of Example 27 compound (Exemplary Compound No. 1183)
NH
OH
Example 27 compound (Exemplry Compound No. 1183) nmol of Example 21 compound were dissolved in 30 pi of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 pi of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 100 p1 of water were added, and the aqueous layer was washed three times with 100 pI of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 28-100%
CH
3 CN (linear gradient, 10 min); 60 0 2 ml/min; 254 nm), and the fraction at 5.2 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 7.57 minutes. Yield: 16.8 nmol in terms of UV measurement at 260 nm, hmax (H 2 0) 258 nm, ESI-Mass (negative); 1498.5 (Example 28) Synthesis of Example 28 compound (Exemplary Compound No. 1219) S:/Chemical/Sankyo/FP200343/FP200343sl .doc P89346/FP-200343/gds-mg/specification//03.05.05 Example 28 compound (Exemplary Compond No. 1219) 100 nmol of Example 18 compound were dissolved in 100 pi of anhydrous DMF, and 3 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 p.
of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 1p of water were added, and the aqueous layer was washed three times with 200 pl of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 72-100%
CH
3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 3.3 minutes was collected. Yield: 8.6 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 260.5 nm, ESI-Mass (negative); 1791.4 (Example 29) Synthesis of Example 29 compound (Exemplary Compound No. 1220)
NH
2 o N y p d N OH N HN O=P-OH N
N
0=P-OH N 0=P-OH 0 Eame 29 compoud (Exemplary Compound No. 1220) 100 nmol of Example 18 compound were dissolved in 100 pi S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 of anhydrous DMF, and 3 mg of 2-(myristoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 3 il of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 pi of water were added, and the aqueous layer was washed three times with 200 pl of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 52-100%
CH
3 CN (linear gradient, 10 min); 600C; 2 ml/min; 254 nm), and the fraction at 3.7 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 15-100% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 10.96 minutes. Yield: 41.6 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 259.1 nm, ESI-Mass (negative); 1679.5 (Example Synthesis of Example 30 compound (Exemplary Compound No. 1221)
NH,
H IN N1 ample 30 compe o ond (Exemplary Compoud No. 1221) 100 nmol of Example 18 compound were dissolved in 100 pl of anhydrous DMF, and 3 mg of 2-(decanoyloxy)ethyl bromide (Devinsky, Ferdinand et al., Collect. Czech. Chem. Commun. 49, 12, 1984, 2819-2827), and 3 pl of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 p1 of water S:/Chemical/Sankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 were added, and the aqueous layer was washed three times with 200 il of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 34-100% CH3CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 0.1M aqueous triethylamine acetate (TEAA), pH 7; 15-100%
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 8.92 minutes. Yield: 46.6 nmol in terms of UV measurement at 260 nm, Xmax (HzO) 259.3 nm, ESI-Mass (negative); 1566[M-H]-.
(Example 31) Synthesis of Example 31 compound (Exemplary Compound No. 1362) NH2 OH N
OH-OH
&=P-OH
0 O Exampl. 31 ompound (Exmplary Compound No. 1362) Synthesis was carried out using the compound of Example 17 described in Japanese Patent Application (Kokai) No. 2002- 249497 (2.0 pmol) as the 5'-O-DMTr-riboadenosine analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 2, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 3. For the oxidation or sulfurizing agent, iodine was used in cycles 1 and 3, and xanthane hydride (Tokyo Kasei Kogyo) was used in cycle 2.
S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 1 and 3): lodine/water/pyridine/tetrahydrofuran; 15 sec.
sulfurization (cycle Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 30 min.); 40 0 C; 10 ml/min; 254 nm), and the fractions that eluted at 10.9 and 12.0 minutes corresponding to the two diastereomer were collected. The present compound eluted in the vicinity of 8.09 and 8.50 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min). (Yield: 749 nmol as UV measured value at 260 nm) kmax (H 2 0) 258 nm, ESI-Mass (negative): 1104.2 (Example 32) S:/Chemical/Sankyo/FP200343/FP200343s1.doc P89346/FP-200343/gds-mg/specification//03.05.05 Synthesis of Example 32 compound (Exemplary Compound No. 1363)
NH,
OHN NH,
~WN
OH N Example 32 compound (Exemplary Compound No. 1363) Synthesis was carried out using the compound of Example 17 described in Japanese Patent Application (Kokai) No. 2002- 249497 (2.0 pmol) as the 5'-O-DMTr-riboadenosine analog bound to a CPG support with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycle 1, 5'-DMT-3'-(O-methyl) adenosine(N-bz)2'-phosphoramidite (ChemGene) was used in cycle 2, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 3. For the oxidation or sulfurizing agent, iodine was used in cycles 1 and 3, and xanthane hydride (Tokyo Kasei Kogyo) was used in cycle 2.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 1 and 3): lodine/water/pyridine/tetrahydrofuran; 15 sec.
sulfurization (cycle Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been S:/ChemicaUSankyo/FP200343/FP20043sl .doc P89346/FP-200343/gds-mg/specification/03.50 removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the pH was adjusted to 2.0 by adding aqueous hydrochloric acid (2 N) to the remaining residue followed by reacting for 5 hours at 30 0 C to remove the silyl group. After neutralizing with aqueous ammonia and distilling off the solvent, the product was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (GL Science Inertsil Prep-ODS x 250 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 30 min.); 40 0 C; 10 ml/min; 254 nm), and the fractions that eluted at 11.5 and 12.7 minutes corresponding to the two diastereomers were collected. The present compound eluted in the vicinity of 8.48 and 8.97 minutes when analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-
CH
3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min). (Yield: 555 nmol as UV measured value at 260 nm) Amax (H 2 0) 258 nm, ESI-Mass (negative): 1118.2 (Example 33) Synthesis of Example 33 compound (Exemplary Compound No. 1369)
NHN
HO\i ^OH NH N
NN
O =P-O H N O OH Example 33 compound (Exempl y Compound No. 1369) 100 nmol of Example 2 compound were dissolved in 50 i1 of anhydrous DMF, and 2 pg of 2-bromoethanol (Tokyo Kasei Kogyo), S:/ChemicaVSankyo/FP200343/FP200343s 1.doc P89346/FP-200343/gds-mg/specification//03050 154 and 2 p1 of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 200 p1 of water were added, and the aqueous layer was washed three times with 200 p1 of AcOEt.
After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-25% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 6.4 minutes was collected.
When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-15% CH 3 CN (linear gradient, min); 60 0 C; 1 ml/min), it eluted at 12.54 minutes. Yield: 20.3 nmol in terms of UV measurement at 260 nm, Amax (H 2 0) 258.1 nm, ESI-Mass (negative); 1118.2[M-H]-.
(Example 34) Synthesis of Example 34 compound (Exemplary Compound No. 1394)
NH,
ON NH, O=P-OH W N G =-OH N 00 OH Example 34 compound (Exemplary Compound No. 1394) 100 nmol of Example 19 compound were dissolved in 50 p1 of anhydrous DMF, and 2 pi of 2-bromoethanol (Tokyo Kasei Kogyo), and 2 p1 of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 200 p of water were added, and the aqueous layer was washed three times with 200 p of AcOEt.
After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck S:/Chemical/Sankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-25% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min; 254 nm), and the fraction at 6.0 minutes was collected.
When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 0-15% CH3CN (linear gradient, min); 60 0 C; 1 ml/min), it eluted at 11.60 minutes. Yield: 48.2 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258.0 nm, ESI-Mass (negative); 1242.2 (Example Synthesis of Example 35 compound (Exemplary Compound No. 1645)
NH,
on N S=P-OH
N
OH
O
S=P-OH
Exampl 35 compound SH (ExempMy Compound No. 1645) Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-Phosphate CPG (Glen Research) (0.5 xnol) was used as the solid phase carrier. The compound of Example 16 described in Japanese Patent Application (Kokai) No. 2002-249497 was used as the phosphoramidite in cycle 1, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used in cycle 2, 3'-(O-methyl) adenosine(N-bz)2'-phosphoramidite (ChemGene) was used in cycle 3, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1, 2 and 3, and iodine was used in cycle 4.
Condensation Cycle: S:/ChemicalSankyo/FP200343/FP200343s l.doc P89346/FP-200343/gds-mg/specification//03.05.05 156 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, c- tetrazole/acetonitrile; 10 to 20 min.
eC 3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
00 4) Oxidation (cycle lodine/water/pyridine/tetrahydrofuran; r- VI) 15 sec.
00 sulfurization (cycles 1, 2 and Xanthane hydride (0.02 M)/ N^ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the remaining residue was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3 CN (linear gradient, 10 min.); 60 0 C; 2 ml/min), and the fractions that eluted at 4.8-5.2 minutes corresponding to the four diastereomers were collected.
After the solvent was evaporated under reduced pressure, 1 ml of aqueous hydrochloric acid (0.01N) was added to the remaining residue to accurately adjust the pH to 2.0, followed by reaction at 30 0 C for 5 hours to remove the DMTr group and the silyl group. After neutralization with aqueous ammonia, the deprotected silanol and DMTrOH were removed by extraction with ethyl acetate to obtain the desired compound. When the present compound was analyzed by reverse phase HPLC (column (Merck chromolith (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-25% CH 3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), it eluted at 4.72 and 5.06 minutes. Yield: 70 nmol (in terms of UV measurement at 260 nm), Xmax (H 2 0) 258 nm, ESI- Mass (negative); 1230.1 P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 (Example 36) Synthesis of Example 36 compound (Exemplary Compound No. 1646) NHn,
N
(emplary C d N. 1?6) 0=P-OH W N HNH2 Example 36 compound SH (Eoeoiptory Compound No. 1646) Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-Phosphate CPG (Glen Research) (0.5 pmol) was used as the solid phase carrier. The compound of Example 16 described in Japanese Patent Application (Kokai) No. 2002-249497 was used as the phosphoramidite in cycle 1, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used in cycle 2, adenosine(N-bz)2'-phosphoramidite (ChemGene) was used in cycle 3, and the compound of Example 8a described in Japanese Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1 and 2, and iodine was used in cycles 3 and 4.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 3 and 4): lodine/water/pyridine/tetrahydrofuran; 15 sec.
sulfurization (cycles 1 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
S:/Chemical/Sankyo/FP200343/FP00343s 1. doc P89346/FP-200343/gds-mg/specification/03.05.05 After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the remaining residue was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3 CN (linear gradient, 10 min.); 60 0 C; 2 ml/min), and the fractions that eluted at 4.7-5.0 minutes corresponding to the two diastereomers were collected.
After the solvent was evaporated under reduced pressure, 1 ml of aqueous hydrochloric acid (0.01N) was added to the remaining residue to accurately adjust the pH to 2.0, followed by reaction at 30 0 C for 5 hours to remove the DMTr group and the silyl group. After neutralization with aqueous ammonia, the deprotected silanol and DMTrOH were removed by extraction with ethyl acetate to obtain the desired compound. When the present compound was analyzed by reverse phase HPLC (column (Merck chromolith (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-20% CH 3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), it eluted at 4.12 and 4.44 minutes. Yield: 95 nmol (in terms of UV measurement at 260 nm), Amax (H 2 0) 258 nm, ESI- Mass (negative); 1214.2 (Example 37) Synthesis of Example 37 compound (Exemplary Compound No. 1648) S :/ChemicaUSankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mg/specification/03.05.05 159
NH
2
N
oo OH NH
S=P-OH
NH
OH0
N
SP-OH
o-s Exampl 37 compound (Exemplary Compound No. 1648) nmol of Example 35 compound were dissolved in 40 p1 of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 i of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 il of water were added, and the aqueous layer was washed three times with 200 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3
CN
(linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 6.4 minutes was collected. Yield: 12.6 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 258 nm, ESI-Mass (negative); 1568.3 (Example 38) Synthesis of Example 38 compound (Exemplary Compound No. 1649) Example 38 compound (Exemplary Compound No. 1649) S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 nmol of Example 36 compound were dissolved in 40 .l of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 1l of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 .l of water were added, and the aqueous layer was washed three times with 200 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3
CN
(linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 6.4 minutes was collected. Yield: 21.8 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 258 nm, ESI-Mass (negative); 1552.3 (Example 39) Synthesis of Example 39 compound (Exemplary Compound No. 1651) CM s Eaple 39 compound (Exemplary Compound No. 1651) nmol of Example 35 compound were dissolved in 40 .l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 p.
of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 .1 of water were added, and the aqueous layer was washed three times with 200 .l of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC S :/Chemical/Sankyo/FP200343/FP200343s 1 .doc P89346/FP-200343/gds-mg/specification//03.05.05 (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100%
CH
3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 6.7 minutes was collected. Yield: 11.4 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258 nm, ESI-Mass (negative); 1540.3 (Example Synthesis of Example 40 compound (Exemplary Compound No. 1652) N NH 14-0 0 14 0H, O- N N 0=P-OH N N O HO c6
S=P-OH
O=P-OH
Example 40 compound (Exemply Compound No. 1652) nmol of Example 36 compound were dissolved in 40 jil of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 ±i of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 .1 of water were added, and the aqueous layer was washed three times with 200 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100%
CH
3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 7.0 minutes was collected. Yield: 23.1 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 258 nm, ESI-Mass (negative); 1524.3 (Example 41) S :/ChemicaUSankyo/FP200343/FP200343s L~doc P89346/FP-200343/gds-mglspcification//03.05.05 Synthesis of Example 41 compound (Exemplary Compound No. 1663) 6H NH2 HO OH^
NH,
S=P-OH N N
NH,
00 OH 0 8=P-OH N N 00 Example 41 compound 9 S(Exemplary Compound No. 1683) O=P-OH S H Synthesis was carried out with the DNA synthesizer based C on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-Phosphate CPG (Glen Research) (0.5 muol) was used as the solid phase carrier. The compound of Example 14 described in Japanese Patent No. 3420984 was used as the phosphoramidite in cycle 1, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used in cycle 2, 5'-DMT-3'-(O-methyl) adenosine(N-bz)2'-phosphoramidite (ChemGene) was used in cycle 3, and the compound of Example 8a described in Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1, 2 and 3, and iodine was used in cycle 4.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycle lodine/water/pyridine/tetrahydrofuran; sec.
sulfurization (cycles 1, 2 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure, and the pH was accurately adjusted to by adding 1 ml of aqueous hydrochloric acid (0.01 N) to the remaining residue, followed by reacting for 5 hours at 30 0 C to remove the DMTr group and the silyl group. After neutralizing with aqueous ammonia, the deprotected silanol and the DMTrOH were removed by extraction with ethyl acetate to obtain the desired compound. The product eluted at 3.75, 4.12, 4.53 and 4.76 minutes when analyzed by reverse phase HPLC (column (Merck chromolith (4.6 x 50 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 5-20% CH 3 CN (linear gradient, 10 min.); 60°C; 2 ml/min). Yield: 111 nmol (in terms of UV measurement at 260 nm), Xmax (H 2 0) 258 nm, ESI-Mass (negative); 1216.1 (Example 42) Synthesis of Example 42 compound (Exemplary Compound No. 1664)
NX,
0HO OO NH 2 P-OH I 0=P-OH Exampl 42 compond SM (Exemplary Compound No. 1684) Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) to 4) using a synthesis program for ordinary synthesis of 1 pmol of RNA. 3'-Phosphate CPG (Glen Research) (0.5 pmol) was used as the solid phase carrier. The compound of Example 14 described in Japanese Patent No. 3420984 was used as the phosphoramidite in cycle 1, 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used in cycle 2, 5'-DMT-3'-(O-methyl) S:/ChemicalSankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 adenosine(N-bz)2'-phosphoramidite (ChemGene) was used in cycle 3, and the compound of Example 8a described in Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1 and 2, and iodine was used in cycles 3 and 4.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycles 3 and 4): lodine/water/pyridine/tetrahydrofuran; 15 sec.
sulfurization (cycles 1 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the remaining residue was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3 CN (linear gradient, 10 min.); 60 0 C; 2 ml/min), and the fractions that eluted at 4.6-4.9 minutes corresponding to the two diastereomers were collected.
After the solvent was distilled off under reduced pressure, 1 ml of aqueous hydrochloric acid (0.01N) was added to the remaining residue to accurately adjust the pH to followed by reaction at 30 0 C for 5 hours to remove the DMTr group and the silyl group. After neutralization with aqueous ammonia, the deprotected silanol and DMTrOH were removed by S :/ChemicalSankyo/FP200343/FP200343s L~doc P89346/FP.200343/gds-mg/specification/03.05.05 165 extraction with ethyl acetate to obtain the desired compound.
When the present compound was analyzed by reverse phase HPLC (column (Merck chromolith (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-20% CH 3 CN (linear gradient, min); 60 0 C; 2 ml/min), it eluted at 2.40 and 3.01 minutes.
Yield: 127 nmol (in terms of UV measurement at 260 nm), Xmax
(H
2 0) 258 nm, ESI-Mass (negative); 1200.15 (Example 43) Synthesis of Example 43 compound (Exemplary Compound No. 1666)
O
Example 43 compound (Exemplary Compound No. 1686) nmol of Example 41 compound were dissolved in 40 p1 of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 p. of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 p.
of water were added, and the aqueous layer was washed three times with 200 .1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3
CN
(linear gradient, 8 min); 60 0 2 ml/min), and the fraction at 6.9 minutes was collected. Yield: 9.5 nmol in terms of UV measurement at 260 nm, Xmax (H20) 258 nm, ESI-Mass (negative); 1554.3 S:/ChemicalSankyo/FP200343/FP200343s L~doc P89346/FP.200343/gds-mg/specification//03.0.05 (Example 44) Synthesis of Example 44 compound (Exemplary Compound No. 1667)
NH
2 o C w O N NH 2 0 -OH
S=P-OH
0= OH Example compund (Exe mp ouy Com nd No. 1667) nmol of Example 42 compound were dissolved in 40 il of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 pL of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 il of water were added, and the aqueous layer was washed three times with 200 pg of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3
CN
(linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 6.9 minutes was collected. Yield: 4.6 nmol in terms of UV measurement at 260 nm, mjax (H 2 0) 258 nm, ESI-Mass (negative); 1538.3 (Example Synthesis of Example 45 compound (Exemplary Compound No. 1669) S :/ChemicaUSankyo/FP200343/FP200343s I.doc P89346/FP-200343/gds-mg/specification//03.05.O5 Example 45 compound (Exemplay Compound No. 1669) nmol of Example 41 compound were dissolved in 100 .1 of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 .l of pyridine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 500 .1 of water were added, and the aqueous layer was washed three times with 500 .1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3
CN
(linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 6.6 minutes was collected. Yield: 40.4 nmol in terms of UV measurement at 260 nm, Xmax (H20) 258 nm, ESI-Mass (negative); 1526.3 (Example 46) Synthesis of Example 46 compound (Exemplary Compound No. 1670) S:/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mg/specification//03.05.05 168 Example 46 compound (Exemplary Compound No. 1670) nmol of Example 42 compound were dissolved in 100 1l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 pl of pyridine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 500 p1 of water were added, and the aqueous layer was washed three times with 500 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3
CN
(linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 6.6 minutes was collected. Yield: 41.9 nmol in terms of UV measurement at 260 nm, kmax (H 2 0) 259 nm, ESI-Mass (negative); 1510.29 (Example 47) Synthesis of Example 47 compound (Exemplary Compound No. 1690) Example 47 compound (Exemplary Comound No. 1690) S :/Chemical/Sankyo/FP200343/FP00343s 1. doc P89346/FP-200343/gds-mg/specification//03.05.5 169 Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) c to 4) using a synthesis program for ordinary synthesis of 1 mol (C of RNA. 3'-Phosphate CPG (Glen Research) (0.5 pmol) was used as the solid phase carrier. 5'-DMT-3'-(O-methyl) Adenosine(N- 00 bz)2'-phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1 and 3, 3'-tBDSilyl-ribo Adenosine (N-bz) 00 phosphoramidite (ChemGene) was used in cycle 2, and the compound Cr of Example 8a described in Patent Application (Kokai) No. Hei 0 11-246592 was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1, 2 and 3, and iodine was used in cycle 4.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycle lodine/water/pyridine/tetrahydrofuran; sec.
sulfurization (cycles 1, 2 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure and the remaining residue was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column ((Merck chromolith (4.6 x 50 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3 CN (linear gradient, 10 min.); 60 0 C; 2 ml/min), and the fractions that eluted at 4.7-5.1 minutes corresponding to the two diastereomers were collected.
P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 After the solvent was evaporated under reduced pressure, 1 ml of aqueous hydrochloric acid (0.01N) was added to the remaining residue to accurately adjust the pH to 2.0, followed by reaction at 30 0 C for 5 hours to remove the DMTr group and the silyl group. After neutralization with aqueous ammonia, the deprotected silanol and DMTrOH were removed by extraction with ethyl acetate to obtain the desired compound. When the present compound was analyzed by reverse phase HPLC (column (Merck chromolith (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-20% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min), it eluted at 3.67, 4.01, 4.15 and 4.55 minutes. Yield: 140 nmol (in terms of UV measurement at 260 nm), Amax (H 2 0) 258 nm, ESI-Mass (negative); 1204.1 (Example 48) Synthesis of Example 48 compound (Exemplary Compound No. 1691) >~NH2 OH N
SP-OH
0-OH Example 48 compound (Exemplary Compound No. 1691) nmol of Example 47 compound were dissolved in 40 p1 of anhydrous DMF, and 1 mg of 2-(2,2-dimethyloctadecanoyloxy)ethyl bromide described in Example 22, and 1 p1 of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 il of water were added, and the aqueous layer was washed three times with 200 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH 3
CN
S:/Chemical/Sankyo/FP200343/FP200343s 1.doc P89346/FP-200343/gds-mg/specification//03.05.05 (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at minutes was collected. Yield: 2.5 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 258 nm, ESI-Mass (negative); 1542.3 (Example 49) Synthesis of Example 49 compound (Exemplary Compound No. 1692)
N,
P-OH N HO- ,041
NH
H N N 0 0P-OH Example 49 compound (Exemplary Compound No. 1692) nmol of Example 47 compound were dissolved in 40 pg of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 pg of triethylamine were added thereto, followed by reacting the mixture at room temperature overnight. After completion of the reaction, 300 pl of water were added, and the aqueous layer was washed three times with 200 p1 of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH7; 24-100%
CH
3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 6.8 minutes was collected. Yield: 29.6 nmol in terms of UV measurement 'at 260 nm, kmax (H 2 0) 258 nm, ESI-Mass (negative); 1514.3 (Example Synthesis of Example 50 compound (Exemplary Compound No. 1929)
HOC
2 H40-P(=0) (OH)-K2 -P (OH) (OH)-K 2 1
(OH)-L
1 P (OH) -LI-p-G e p -Aep G e p-A e -p-C e -p-C-Cn-p-T n -p-G n -p-A-p-A n S:/Chemical/Sankyo/FP200343/FP200343s 1 .doc P89346/FP-200343/gds-mg/specification//03.05.05 p-Cn-p-An-p-Gn-p-Tn-p-Te-p-Ge-p-Ae-p-Te-p-Ce-hp A 2-5A analog having the desired sequence was synthesized by coupling various phosphoramidites in order based on one condensation cycle consisting of the following steps 1) to 4) using a DNA synthesizer, a synthesis program for ordinary synthesis of 1 pmol of RNA, and 1 pmol of the compound described in Example 12b of Patent Application (Kokai) No. Hei 7-87982 as the solid phase support.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (25 eq), acetonitrile/tetrazole; to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation: lodine/water/pyridine/tetrahydrofuran; 15 sec.
As the phosphoramidites used to synthesize the antisense oligonucleotide portion, adenine (dAz) phosphoramidite, guanine (dGibu) phosphoramidite, cytosine (dCbz) phosphoramidite, and thymine phosphoramidite (Applied Biosystems) were used for the sequences equivalent to natural type nucleotides, while the compounds of Examples 14, 27, 22 and 9 described in Japanese Patent No. 3420984 were used for the sequences equivalent to non-natural type nucleotides (Ae, Ge, Ce, Te). DMT-butanol-CED phosphoramidite (ChemGene) was used for the phosphoramidite equivalent to L 1 and 5'-DMT-3'- (O-methyl)adenosine(N-bz)2'phosphoramidite (ChemGene), tBDsilyl-riboadenosine(Nbz)phosphoramidite (ChemGene), DMT-3'-(O-methyl)adenosine(Nbz)2'-phosphoramidite (ChemGene), and the phosphoramidite of Example 8a described in Patent Application (Kokai) No. Hei 11- 246592, were coupled in order.
After synthesizing the protected 2-5A analog having the desired structure in the state in which the 5'-DMTr group has been removed, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the protecting group on the nucleic acid base were removed by treating with a mixture of concentrated S:/Chemical/Sankyo/FP200343/FP200343sl.doc P89346/FP-200343/gds-mg/specification//03.05.05 aqueous ammonia and ethanol The solvent was then distilled off under reduced pressure, and 1 ml of triethylamine trihydrofluoride was added to the residue followed by stirring at room temperature. After 24 hours, 200 il of H 2 0 were added, followed by the addition of 10 ml of 1-butanol, allowing to stand for 1 hour at -20 0 C, and centrifuging to obtain a pelletlike precipitate. After gently washing this pellet with EtOH, it was dissolved in 150 il of H 2 0 and then subjected to electrophoresis on 15% denatured acrylamide gel (Ix TBE solution (7 M urea, 0.89 M Tris, boric acid, EDTA solution (pH 8.3, Takara Shuzo), 600 V, 60 minutes). The band that absorbed UV in the gel was cut out and eluted from the gel with 1 ml of an elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH 0.1% SDS). The remaining gel was filtered off, 4 ml of EtOH were added to the filtrate, which was then allowed to stand for 1 hour at -20 0 C followed by centrifugation to obtain a pellet-like precipitate. This was then purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck Chromolith (4.6 x 50 0.1 M aqueous triethyl amine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 8 min.); 60 0 C; 2 ml/min), and the fraction that eluted at 4.02 minutes was collected. The yield was 16.0 nmol (as UV measured value at 260 nm), and Xmax (H 2 0) 259 nm.
(Example 51) Synthesis of Example 51 compound (Exemplary Compound No. 1930)
HOC
2 H40-P(=O) (OH) -K 2 -1-P (OH) -K 1 1 (OH) -K 2 -P (OH)-Li- P (OH) -L-p-T e -p-C e -p-T e -p-T e -p-G e -p-G-p-T n -p-T n -p-G n -p-T n -p-A n p-An-p-G n -p-A n -p-G n -p-A e -p-G e -p-A e -p-p-G e -p-A e -hp The title compound was obtained according to a similar method to Example 50. The present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 3.61 minutes was collected. Yield: S:/Chemical/Sankyo/FP200343/FP200343sl.doc P89346/FP-200343/gds-mg/specification//03.05.05 15.8 nmol (in terms of UV measurement at 260 nm), kmax (H 2 0) 257 nm.
(Example 52) Synthesis of Example 52 compound (Exemplary compound No. 1931)
HOC
2 H40-P (OH) -K 2 P (OH)-K 1 (OH) -K 2 1 -P (OH) -Li- P (OH) -L-p-Tep-Te-p-Ce-p-Ae-p-Ge-p-Gn-p-Cn-p-Cn-p-Tn-p-Cn-p-C n p-An-p-Tn-p-An-p-Tn-p-Ge-p-Ge-pp-Ae-Aep-T e-hp The title compound was obtained according to a similar method to Example 50. The present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 3.66 minutes was collected. Yield: 7.8 nmol (in terms of UV measurement at 260 nm), Xmax (H 2 0) 259 nm.
(Example 53) Synthesis of Example 53 compound (Exemplary Compound No. 1932)
HOC
2
H
4 0-P (OH) -K2--P (OH)-K2--P (OH) -Li- P (OH) -L-p-Ge-p-Te-p-Te-p-Ce-p-Te-p-Cn-p-Gn-p-Cn-p-Tn-p-Gn-p-G n p-Tn-p-Gn-p-An-p-Gn-p-Te-p-Te-p-Te-p--pe-hp The title compound was obtained according to a similar method to Example 50. The present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 8-12% CH 3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min), and the fraction at 8.0-10.0 minutes was collected.
When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-29% CH 3 CN (linear gradient, min); 60 0 C; 1 ml/min), it eluted at 7.54 minutes. Yield: 37.6 nmol (in terms of UV measurement at 260 nm), kmax (H 2 0) 258.9 nm.
S :/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-mg/specification//03.O5.05 (Example 54) Synthesis of Example 54 compound (Exemplary compound No. 1933)
HOC
2 H40-P(=0) (OH) -K 2 P (OH) -K 1 P (OH) -K 2 1 P (OH) -L 1 P (OH) -L-p-G e -p-A e -p-T e -p-G e -p-G e
A
n -p-A n -p-A n -p-T n -p-C n -p-T n p-Cn-p-Tn-p-Gn-p-Cn-p-Ce-p-Ge-p-Ce-p-Ae-p-Te-hp The title compound was obtained according to a similar method to Example 50. The present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 3.74 minutes was collected. Yield: 131 nmol (in terms of UV measurement at 260 nm), Xmax (H20) 262 nm.
(Example Synthesis of Example 55 compound (Exemplary compound No. 1934)
HOC
2 H40-P(=0) (OH) -K2- 1 -P (OH) -K P (OH) -K 2 P (OH) -Li- P (OH) -Lj-p-Ae-p-Te-p-Ge-p-Ge-p-Ce-p-An-p-Cn-p-Cn-p-Tn-p-Cn-p-T n p-Tn-p-Gn-p-Tn-p-Gn-p-Ge-p-Ae-p-Ce-p-Ce-p-Ae-hp The title compound was obtained according to a similar method to Example 50. The present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 3.83 minutes was collected. Yield: nmol (in terms of UV measurement at 260 nm), Xmax (H 2 0) 261 nm.
(Example 56) Synthesis of Example 56 compound (Exemplary Compound No. 1935)
HOC
2 H40-P(=0) (OH) -K2- -P (OH) -K -P (OH) -K P (OH) -Li- P (OH) -L1-p-C e -p-A e -p-G e -p-C e -p-C e -p-A n -p-T n -p-G n -p-G n -p-T n -p-C n p-Cn-p-C n -p-C n -p-C n -p-C e -p-C e -p-C e -p-A e -p-A e -hp The title compound was obtained according to a similar method to Example 50. The present compound was purified by S:/ChemicalSankyo/FP200343/FP200343sl.doc P89346/FP-200343/gds-mg/specification//03.05.05 176 reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 9-25% CH 3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), and the fraction at 3.25 minutes was collected. Yield: nmol (in terms of UV measurement at 260 nm), Xmax (H20) 266 nm.
(Example 57) Synthesis of Example 57 compound (Exemplary compound No. 1936)
HOC
2
H
4 (OH) -K 2 1 -P (OH) -K 1 1 -P (OH) -K 2 1 P (OH) -L 2 -p- Ge-p-TeTe--p-Te-p-e_ epnGn-p-Cn-p-Tn-p-Gn-p-Gn-p-Tnp_-Gnp-A n p-Gn-p-Te-p-Te-p-Te-p-Ce-p-Ae_hp The title compound was obtained according to a similar method to Example 50. Here, Spacer phosphoramidite 18 (Glen Research Inc.) was used as the phosphoramidite corresponding to
L
2 The present compound was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 8-12%
CH
3 CN (linear gradient, 10 min); 60 0 C; 2 ml/min), and the fraction at 8.0-10.0 minutes was collected. When the present compound was analyzed by reverse phase HPLC (column (Tosoh superODS (4.6 x 50 0.1M aqueous triethylamine acetate (TEAA), pH 7; 5-29% CH 3 CN (linear gradient, 10 min); 60 0 C; 1 ml/min), it eluted at 7.52 minutes. Yield: 59.5 nmol (in terms of UV measurement at 260 nm), Xmax (H 2 0) 258.6 nm.
(Example 58) Synthesis of Example 58 compound (Exemplary Compound No. 1103) S :/Chemical/Sankyo/FP200343/FP00343s L~doc P89346/FP-200343/gds-g/specification//3O.05.05 177 OH NH 2 SP-OH N CO NH2 CH 0 N S =P-OH N N 00 o OH 0 Example 58 compound 0=P-OH (Exemplay Compound No. 1103) SH 00 Synthesis was carried out with the DNA synthesizer based on one condensation cycle consisting of the following steps 1) Cq to 4) using a synthesis program for ordinary synthesis of 1 pnol of RNA. 3'-Phosphate CPG (Glen Research) (0.2 mol) was used as the solid phase carrier. 3'-tBDSilyl-ribo Adenosine (N-bz) phosphoramidite (ChemGene) was used as the phosphoramidite in cycles 1, 2 and 3, and the compound of Example 8a described in Patent Application (Kokai) No. Hei 11-246592 was used in cycle 4. For the oxidation or sulfurizing agent, xanthane hydride (Tokyo Kasei Kogyo) was used in cycles 1, 2 and 3, and iodine was used in cycle 4.
Condensation Cycle: 1) Detritylation: Trichloroacetic acid/dichloromethane; 85 sec.
2) Coupling: Phosphoramidite (about 25 eq)/acetonitrile, tetrazole/acetonitrile; 10 to 20 min.
3) Capping: 1-methylimidazole/tetrahydrofuran, acetic anhydride/pyridine/tetrahydrofuran; 15 sec.
4) Oxidation (cycle lodine/water/pyridine/tetrahydrofuran; sec.
sulfurization (cycles 1, 2 and Xanthane hydride (0.02 M)/ acetonitrile-pyridine (9:1 mixed solvent); 15 min.
After synthesizing the 2-5A analog having the desired structure in the state in which the 5'-DMTr group is retained, together with cleaving the oligomer from the support, the cyanoethyl group serving as the protecting group on the phosphorus atom and the benzoyl group on the adenine base were removed by treating with a mixture of concentrated aqueous ammonia and ethanol The solvent was then distilled off P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05 under reduced pressure and the remaining residue was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column ((Merck chromolith (4.6 x 50 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH3CN (linear gradient, 8 min.); 60 0 C; 2 ml/min), and the fractions that eluted at 6.0-6.7 minutes corresponding to the four diastereomers were collected.
After the solvent was evaporated under reduced pressure, 1 ml of aqueous hydrochloric acid (0.01N) was added to the remaining residue to accurately adjust the pH to 2.0, followed by reaction at 30 0 C for 5 hours to remove the DMTr group and the silyl group. After neutralization with aqueous ammonia, the deprotected silanol and DMTrOH were removed by extraction with ethyl acetate. When the remaining aqueous solution was analyzed by reverse phase HPLC (column (((Merck chromolith (4.6 x 0.1M aqueous triethylamine acetate (TEAA), pH7; 0-20%
CH
3 CN (linear gradient, 8 min); 60 0 C; 2 ml/min), the fractions that eluted at 4.6 and 5.4 minutes were collected to obtain the desired compound. Yield: 38 nmol (in terms of UV measurement at 260 nm), kmax (H 2 0) 259 nm (Example 59) Synthesis of Example 59 compound (Exemplary Compound No. 1195) HD2
SN
S=P-OH N N o=6
H,
OHO
S- OH OH 9 Example 59 ompound (Exemplary Compound No. 1195) nmol of Example 58 compound were dissolved in 100 l of anhydrous DMF, and 1 mg of 2-(stearoyloxy)ethyl bromide (Ackerman et al., J. Am. Chem. Soc., 78, 1956, 6025), and 1 .l of pyridine were added thereto, followed by reacting the mixture S:/Chemical/Sankyo/FP200343/FP200343s .doc P89346/FP-200343/gds-mg/specification//03.05.05 at room temperature overnight. After completion of the reaction, 500 p1 of water were added, and the aqueous layer was washed three times with 500 il of AcOEt. After the aqueous layer was evaporated, it was purified by reverse phase HPLC (Shimadzu Seisakusho LC-VP; column (Merck chromolith (4.6 x 50 mm); 0.1M aqueous triethylamine acetate (TEAA), pH 7; 24-100% CH3CN (linear gradient, 8 min); 600C; 2 ml/min), and the fraction at 6.8 minutes was collected. Yield: 8.9 nmol in terms of UV measurement at 260 nm, Xmax (H 2 0) 259 nm.
(Test Example 1) Measurement of Cytotoxic Activity of 2-5A Analogs (MTT Assay) Human lung cancer cell line A549 cells were plated at a density of 800 cells/200 p in a 96-well plate using RPMI1640 (Gibco BRL) (containing 10% Fetal Bovine Serum (Hyclone)) for the medium followed by culturing overnight in 5% C02 at 370C.
Each 2-5A analog was added to each well so that the final concentration became 10 pM, followed by culturing for 72 hours (3 days). After culturing for 72 hours (3 days), MTT dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide) was added in 50 p aliquots to each well at a MTT/RPMI1640 concentration of 5 mg/ml, followed by additionally culturing for 4 hours.
After 4 hours, the medium was removed and 150 p1 of dimethyl sulfoxide were added to each well. After shaking for 5 minutes, UV absorbance at 540 nm was measured to determine the relative ratio of the number of viable cells of the compound dose group to the number of viable cells of an untreated cell group at 72 hours after addition of the test compound.
The cytotoxic activity during addition of the subject compounds (10 pM) with respect to A549 cells is shown in the graph. In this graph, the natural type 2-5A indicates a 3 mer, with a 5'-monophosphate group having the structure shown below (Imai, J. and Torrence, J. Org.
Chem., 1985, 50(9), 1418-1426).
S :/Chemical/Sankyo/FP200343/FP200343s L~doc P89346/FP-200343/gds-mgspecificationllO3.05.05
NH
2
N
Natural type Cytotoxic Activity on A549 Cells During Addition of Subject Compounds (10 jiM) o 100 4 0 a) 4-1 o W 0 K z X4 <Ile
N
As is clear from this graph, during addition to the medium at a concentration of 10 p.M, in contrast to natural type not demonstrating any cytotoxic effects against human lung cancer cell line A549 cells, the subject compounds demonstrated superior cytotoxic activity.
(Test Example 2) S:/Chemical/Sankyo/FP200343IFP200343s 1 doc P89346/FP-200343/gds-mg/specification/lO3.05.05 Measurement of Cytotoxic Activity of 2-5A Analogs (MTT Assay) Human lung cancer cell line A549 cells were plated at a density of 800 cells/200 1l in a 96-well plate using RPMI1640 (Gibco BRL) (containing 10% Fetal Bovine Serum (Hyclone)) for the medium followed by culturing overnight in 5% CO 2 at 37 0
C.
Each 2-5A analog was added to each well so that the final concentration became 0.001-10 pM, followed by culturing for 72 hours (3 days) (n=3 or After culturing for 72 hours (3 days), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) was added in 50 .l aliquots to each well at a MTT/RPMI1640 concentration of 5 mg/ml, followed by additionally culturing for 4 hours. After 4 hours, the medium was removed and 150 p1 of dimethyl sulfoxide were added to each well. After shaking for 5 minutes, UV absorbance at 540 nm was measured to determine the relative ratio of the number of viable cells of the compound dose group to thenumber of viable cells of an untreated cell group followed by calculation of the ICso concentration that inhibits cell growth by The table shows the 50% growth inhibitory concentrations of the subject compounds with respect to A549 cells.
[Table] 50% Growth inhibitory concentrations of the subject compounds with respect to A549 cells Experiment Experiment Experiment Experiment 1 2 3 4 Example 2 1.53 0.91 Example 4 0.48 0.61 0.38 Example 5 0.40 0.32 Example 8 2.23 Example 9 1.39 Example 13 2.54 Example 14 2.34 Example 15 0.061 0.073 Example 16 0.09 Example 17 0.35 Example 19 13 Example 22 0.33 Example 23 0.13 0.091 S :/ChemicaUSankyo/FP200343/FP200343s L~doc P89346/FP-200343/gds-mg/specification//0305.05 182 Example 24 0.30 Example 25 0.91 Example 26 0.15 Example 27 0.36 C< Example 28 0.13 Example 29 0.14 00 Example 30 0.43 r- Example 33 2.15 In Example 34 6.60 00 -c As is clear from the above table, in contrast to natural Stype 2-5A not demonstrating any cytotoxic effects against human lung cancer cell line A549 cells even at 10 pM, the subject compounds demonstrated superior cytocidal activity.
[Industrial applicability] The compounds of the present invention have stability and excellent activity (particularly antitumor activity), and are useful as pharmaceutical drugs (particularly antitumor agents).
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
P:\OPER\PDB\Speci\2003284578 Ispa.doc 7/7/05

Claims (21)

1. A 2 ',5'-oligoadenylate analog represented by the 00 O general formula 0 0 0 /O 0 00 11 7 E -P-E P-E R P- R 2 13 14 15 I R
2 R 3 R R R 00 m n S[wherein m represents 0; n represents 0 or 1; R' represents an 03 alkoxy group having from 1 to 6 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, an alkylthio group having from 1 to 4 carbon atoms which may be substituted, an amino group, an amino group protected by a nucleic acid synthesis protecting group, an amino group substituted by alkyl group(s) having from 1 to 6 carbon atoms which may be substituted, an alkyl group having from 1 to 6 carbon atoms which may be substituted, an aryloxy group which may be substituted, or an arylthio group which may be substituted, or a group of formula: X 1 -X 2 -X 3 R 2 R 3 R 4 R 5 and R 6 represent a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, an alkoxy group having from 1 to 4 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, an alkylthio group having from 1 to 4 carbon atoms which may be substituted, or a group of formula: X 1 -X 2 -X 3 R 7 represents an oxygen atom, a sulfur atom, a -O(CH 2 CH 2 0)q- group (q represents an integer of 2 to or an oxyalkyleneoxy group having from 1 to 6 carbon atoms; R 8 represents a hydrogen atom, an alkyl group having from 1 to 6 carbon atoms which may be substituted, an aralkyl group which may be substituted, an aryl group which may be substituted, or a 5 '-phosphorylated oligonucleotide analog which has one hydroxyl group removed from the 5'-phosphoric acid group; E 1 represents K 2 E 2 represents K 1 E represents K 2 or K 3 and E 4 represents K 1 K 2 K 3 or K 4 (K 1 K2, S:/ChemicallSankyo/Fp0343/FP0343aucI I.doc P$9346/FP-0343/acamendd claims for au/0.02.07 (K 3 and K 4 represent OH O ODOO 0 00 00 K 1 K 2 K 3 K 4 r K K2 K K4 respectively, wherein, B represents a purin-9-yl group or a 00 substituted purin-9-yl group having substituent(s) selected from the following Group a, A represents an alkylene group having C. from 1 to 4 carbon atoms, D represents an alkyl group having from 1 to 6 carbon atoms which may be substituted, or an alkenyl group having from 2 to 6 carbon atoms which may be substituted); Xi represents an alkyl group having from 1 to 24 carbon atoms which may be substituted, or an aryl group which may be substituted, or an aralkyl group which may be substituted; X 2 represents a -NHC(=O)NH-, or a group; and X 3 represents an alkylene group having from 1 to 6 carbon atoms which may be substituted] (provided that compounds in which n is 1, R 2 R 3 R 4 and R 6 are a hydroxyl group, R 7 is an oxygen atom, and R 8 is a 2- hydroxyethyl group are excluded), or a pharmacologically acceptable salt thereof. (Group a) a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, an alkoxy group having from 1 to 6 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, an alkylthio group having from 1 to 4 carbon atoms which may be substituted, an amino group, S:/Chemical/Sankyo/FP0343/FPO343aucI .doc P89346/FP-0343/acfamended claims for au/20.02.07 San amino group protected by a nucleic acid synthesis Sprotecting group, an amino group substituted by alkyl group(s) having from 0 1 to 4 carbon atoms which may be substituted, an alkyl group having from 1 to 6 carbon atoms which may 00 be substituted, and y- a halogen atom. 00 (N 2. A 2',5'-oligoadenylate analog or pharmacologically O acceptable salt thereof according to Claim 1, wherein R' is an C( alkoxy group having from 1 to 4 carbon atoms which may be substituted, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, or an alkylthio group having from 1 to 4 carbon atoms which may be substituted, or a group of formula: XI-X 2 Xi is an alkyl group having from to 24 carbon atoms which may be substituted; X 2 is a or group; and X 3 is an alkylene group having from 1 to 4 carbon atoms which may have a substituent.
3. A 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to Claim 1, wherein R 7 represents an oxygen atom, a -O(CH 2 CH 2 0)q- group (q represents an integer of 2 to or an oxyalkyleneoxy group having from 1 to 6 carbon atoms; and R 8 is a hydrogen atom, an alkyl group having from 1 to 6 carbon atoms which may be substituted, or a phosphorylated oligonucleotide analog which has one hydroxyl group removed from the 5'-phosphoric acid group.
4. A 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to Claim 1, wherein D is a methyl group or a 2-propenyl group. A 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to Claim 1, wherein E 3 is K 3 and A is a methylene, ethylene, or propylene group.
S:/Chemical/Sankyo/FPO343/FP0343auc I.doc P89346/FP-0343/ac/amended claims for au/20.02.07 S6. A 2',5'-oligoadenylate analog or pharmacologically -n acceptable salt thereof according to Claim 1, wherein B is a 6- 00 0 aminopurin-9-yl (that is, adeninyl), 6-amino-8-bromopurin-9-yl,
6-amino-8-chloropurin-9-yl, 6-amino-8-fluoropurin-9-yl, 6-amino- 00 8-methoxypurin-9-yl, 6-amino-8-ethoxypurin-9-yl, 6-amino-8-t- Sbutoxypurin-9-yl, 6-amino-2-bromopurin-9-yl, 6-amino-2- 00 chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 6-amino-2- Smethoxypurin-9-yl, 6-amino-2-ethoxypurin-9-yl, 6-amino-2-t- butoxypurin-9-yl, or 2,6-diaminopurin-9-yl group.
7. A 2',5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to Claim 1, wherein B is 6- aminopurin-9-yl (that is, adeninyl) or 6-amino-8-bromopurin-9-yl.
8. A pharmaceutical composition comprising a oligoadenylate analog or pharmacologically acceptable salt thereof according to Claims 1 to 7.
9. A 5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to any of Claims 1 to 7, for use as an antiviral drug.
A 5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to any of Claims 1 to 7, for use as an antitumour drug.
11. A 5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to any of Claims 1 to 7, for use as an antisense drug.
12. Use of a 5'-oligoadenylate analog according to any of Claims 1 to 7 in the preparation of an antiviral medicament.
13. Use of a 5'-oligoadenylate analog according to S:/Chemical/Sankyo/FP0343/FP0343aucl .doc P89346/FP-0343/acf/amended claims for au/20.02.07 187 any of Claims 1 to 7 in the preparation of an antitumour Smedicament. 0
14. Use of a 5'-oligoadenylate analog according to any of Claims 1 to 7 in the preparation of an antisense 00 medicament.
15. A method of treating a viral disease, comprising C administration of an effective amount of a Sanalog or pharmacologically acceptable salt thereof according to Cl any of Claims 1 to 7 to a subject in need thereof.
16. A method of treating a tumour, comprising administration of an effective amount of a analog or pharmacologically acceptable salt thereof according to any of Claims 1 to 7 to a subject in need thereof.
17. A method of treating a disease that can be treated by antisense therapy, comprising administration of an effective amount of a 5'-oligoadenylate analog or pharmacologically acceptable salt thereof according to any of Claims 1 to 7 to a subject in need thereof.
18. A 5'-oligoadenylate analog or pharmacologically acceptable salt thereof substantially as hereinbefore described and/or exemplified.
19. A pharmaceutical composition comprising a oligoadenylate analog or pharmacologically acceptable salt thereof substantially as hereinbefore described and/or exemplified.
Use of a 5'-oligoadenylate analog or pharmacologically acceptable salt thereof substantially as hereinbefore described and/or exemplified.
21. A method of treating a viral disease, tumour or S:/Chemical/Sankyo/FPO343/FP0343aucll.doc P89346/FP-0343/acfamended claims for au/20.02.07 disease that can be treated by antisense therapy substantially as hereinbefore described and/or exemplified. 00 0 S:/ChemicallSankya/FP0343/FP0343aucI I .doc P836PO4/a'mcd lisfoauO..0 P893461FP-0343/acf/amended claims for au/20.02.07
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